Breeding resistant wheat (Triticum aestivum) cultivars is the most efficient way to manage wheat stripe rust, a highly destructive disease caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst). Therefore, the exploration of new resistance genes is ongoing. The resistance gene Yr85 confers protection against predominant Pst races in China at all stages of plant development. Here, we fine-mapped Yr85 to a 0.12 cM interval between allele-specific quantitative PCR markers XK1B-61 and XK1B-65, corresponding to a 1.76 Mb region on chromosome 1BS in the IWGSC RefSeq v2.1 reference genome (wheat cultivar Chinese Spring), using 5507 F5 plants derived from heterozygous F4 plants, themselves descendants of an AvS × AvSYr85NIL cross. Haplotype analysis and whole-genome resequencing suggested that Yr85 may be from a distant source. Moreover, agronomic trait evaluation indicated that Yr85 is located in a chromosomal region lacking linkage drag. Genotyping of 309 Chinese wheat cultivars and lines with a marker that cosegregated with Yr85 during fine mapping identified one cultivar possibly carrying the resistance gene. Our findings indicate that Yr85 has potential for use in wheat-breeding programs in China. This study lays the foundation for map-based cloning and marker-assisted selection of Yr85.

The deployment of Yr genes has long been a key strategy in wheat breeding programmes aimed at managing stripe rust caused by Puccinia striiformis. Emergence of new lineages originating from different plant hosts challenges the effectiveness of resistance. The resistance spectra of Yr genes across P. striiformis lineages from various hosts have not previously been explored in Kazakhstan. To address this gap, we genotyped P. striiformis isolates collected from wheat, triticale, barley and durum wheat using the Genobait (GBTS) 20K SNP array. Pathotyping of the isolates was performed using the Yr single-gene set and the Chinese set of wheat lines. Genotyping revealed high genetic diversity in the P. striiformis populations from wheat (Simpson lambda = 0.97), followed by barley (lambda = 0.95) and triticale (lambda = 0.92), while the lowest diversity was observed in the population from durum wheat (lambda = 0.91). High gene flow and low F ST values among the isolates from the different cereal crops except barley suggested high genetic similarity. Genetic group G1 exhibited broad host adaptability, occurring on all four hosts, whereas G2 was mainly restricted to barley with limited overlap with triticale. Although predominant on barley, some G2 isolates also showed virulence to several Yr genes (Yr1, Yr6-9, Yr17, Yr43, Yr44, YrSp and YrExp2). The P. striiformis population in this region lacked virulence against Yr5, Yr10 and Yr15. These findings improve our understanding of the potential risks posed by different host-associated lineages in reshaping the genetic structure of the stripe rust pathogen in Kazakhstan.

Although viruses are primarily characterized as pathogenic agents, certain viruses confer advantages to their hosts. The extent to which a virus can improve host biological performance, however, remains a fascinating topic in virology. An endornavirus, Rhizoctonia solani endornavirus IM (RsEV-IM), was identified as prevalent in Rhizoctonia solani isolates obtained from potato plants. Comparative analysis with a virus-free isogenic strain demonstrated that RsEV-IM infection enhances mycelial growth, sclerotium formation, stress tolerance, and fungal virulence across multiple plant species. Inoculation tests involving numerous R. solani strains confirmed that only RsEV-IM-infected strains exhibited high pathogenicity, independent of other mycovirus infections. Additionally, the secreted protein fraction of RsEV-IM-infected fungus contained elevated levels of various proteins, including those involved in cell wall degradation. This fraction not only facilitated R. solani infection but also suppressed the growth of other fungi and bacteria. These findings position RsEV-IM as a beneficial virus that widely enhances its host’s biological fitness. From both pathological and ecological perspectives, these observations are significant, as they reveal that a mycovirus can serve as a key virulence determinant in fungal populations and potentially shape microbial community dynamics in natural environments.IMPORTANCEFungal pathogenicity and ecological traits have long been thought to be primarily governed by endogenous genetic factors. However, this study reveals a mutualistic relationship between an endornavirus (RsEV-IM) and Rhizoctonia solani, demonstrating that viral infection enhances fungal virulence and ecological fitness. RsEV-IM stimulates fungal growth and the secretion of cell wall-degrading enzymes, resulting in a severe disease phenotype. Ecologically, RsEV-IM-infected fungi potentially gain a competitive advantage over soil microbiota. These findings present a key example of a virus acting as an essential extrachromosomal determinant of fungal pathogenicity and ecosystem interactions. Our results advance the understanding of fungal virulence mechanisms and underscore the broader significance of beneficial virus-fungus associations in agriculture and microbial ecology.

Liquid-liquid phase separation (LLPS) has emerged as an important strategy for plant stress resistance, yet its dynamic regulation during plant-pathogen interaction remains poorly understood. Here, we demonstrate that the stripe rust fungus Puccinia striiformis f. sp. tritici (Pst) deploys effector Hasp170 (one haustorial secreted protein) to subvert wheat immunity by directly disrupting host LLPS. Hasp170 targets the intrinsically disordered region (IDR1) of the wheat nuclear protein TaPSTE (phase separation protein targeted by effector), which forms LLPS-dependent biomolecular condensates. Within these condensates, TaPSTE recruits the transcription factor TaNF-YC, thereby activating the expression of genes driving reactive oxygen species burst and Ca2+ influx, key components for disease resistance. By binding TaPSTE's IDR1, Hasp170 impairs condensate formation and prevents TaNF-YC recruitment, consequently suppressing host immunity and facilitating fungal parasitism. This reveals a virulence strategy where pathogens directly manipulate host biomolecular condensates to evade immune responses.

Plant pathogen evolution is driven by complex biological and ecological processes with major consequences for food security. Understanding how genetic diversity arises and is maintained is critical for predicting disease emergence. The wheat stripe rust fungus Puccinia striiformis f. sp. tritici is among the world's most destructive crop pathogens. As a dikaryotic fungus harboring two distinct haploid nuclei, Puccinia striiformis f. sp. tritici offers an excellent model to investigate nuclear-level evolution. Here, using population genomics of 507 global isolates and haplotype-phased genomes, we reconstruct the evolutionary history of Puccinia striiformis f. sp. tritici and its two nuclei. We show that two ancient nuclear lineages, nuclA and nuclB, originated from a common ancestor and diverged approximately 10,000 years ago, predating modern agriculture. Their differentiation was reinforced during wheat domestication and early agricultural expansion, which imposed new ecological and selective pressures. Subsequent wheat dispersal and regional adaptation promoted the emergence of two allopatric homozygous populations: nuclA-nuclA, which is predominant in south Asia/east Africa, while nuclB-nuclB is predominant in China, with both undergoing sexual recombination. In contrast, later agricultural intensification favored clonal propagation and facilitated recurrent somatic nuclear exchange, giving rise to a globally dominant heterokaryotic population (nuclA-nuclB) with high heterozygosity and broad adaptability. Notably, similar virulence traits can arise independently in clonal populations and sexual populations, resulting in convergent evolutionary outcomes. Together, our results establish a unified evolutionary framework in which host domestication, reproductive strategy, and nuclear-lineage dynamics jointly shape the origin, diversification, and global success of a major crop pathogen.

The HTAP stripe rust resistance gene Yr52 was mapped to a 2.5 8 Mb region and significantly enhanced adult plant stripe rust resistance when combined with QYr.nwafu 6B S .6. Stripe rust is one of the most devastating diseases of wheat. Resistant cultivars are the most economically effective control method. Spring wheat germplasm PI 660057 (carrying Yr52) remained resistant to stripe rust in China. In this study, we developed 145 recombinant inbred lines (RILs) from a cross of PI 660057 and Kenong 9204. The RILs and parental lines were evaluated for stripe rust response across four field environments and genotyped using the GenoBaits WheatSNP16K array. Two stable QTLs from PI 660057 were identified on chromosome arms 6BS (QYr.nwafu-6BS.6) and 7BL (Yr52), with QYr.nwafu-6BS.6 likely being Yr78. The combination of Yr52 and QYr.nwafu-6BS.6 provided better resistance to stripe rust than either gene or QTL alone. To better characterize the genetic and molecular characteristics of Yr52, we utilized a population of 552 residual heterozygous lines (RHLs) to precisely localize Yr52 to a 1.19-cM interval on chromosome 7BL between markers XK7B-16 and XK7B-21. We also developed a co-segregating molecular marker for Yr52 and selected a series of Yr52-bearing lines with superior agronomic traits. These results lay a foundation for the map-based cloning of Yr52 and its subsequent application in gene pyramiding.

Abstract Triadimefon resistance in Puccinia striiformis f. sp. tritici ( Pst ), the fungal pathogen of wheat stripe rust, is increasingly observed in China, but the mechanisms beyond cytochrome P450 14α-demethylase ( Cyp51 ) mutations remain unknown. Through bulked segregant analysis and RNA-seq of a sexual Pst population, we identify three vacuolar iron transporter genes ( CCC1 ), PstCCC1.1 , PstCCC1.2 and PstCCC1.3 , as key triadimefon sensitivity determinants. Functional analyses show that RNAi-mediated silencing of these genes reduce fungicide resistance, while heterologous expression of natural mutants ( PstCCC1.2 R82C,S86P,E109K and PstCCC1.3 R306C,S310P,E333K ) in yeast and Fusarium graminearum reduce cytosolic iron content and thereby increase resistance to triadimefon. Combined microscale thermophoresis (MST) and isothermal titration calorimetry (ITC) show that these two mutants exhibit increased iron affinities, linking specific amino acid changes to the detoxification ability. This iron-mediated resistance mechanism is conserved across fungal species and represents a triazole resistance pathway independent of Cyp51, with implications for managing fungal diseases, especially stripe rust.

Stripe rust, caused by Puccinia striiformis (Ps), is a destructive disease affecting various crops worldwide, including wheat, barley, rye, triticale, and certain graminaceous hosts. Recently, clonal lineages of Ps on wheat have been reported in Uzbekistan, a Central Asian country. However, it is still unclear how these clonal lineages persist throughout the year and whether they are exclusive to wheat or can also infect other hosts. To better understand, we conducted surveillance in various locations of Uzbekistan (Jizzakh, Qashqadaryo, Samarkand, and Sirdaryo) in 2024. Site selection of barley and wild barley fields was done based on their proximity to infected wheat fields. Our results showed low genetic distance (FST) among the wheat, barley, and wild barley Ps populations. The wheat Ps population showed the lowest genotypic diversity (Simpson's diversity = 0.46) while high genotypic diversity was found in barley (0.87) and wild barley (0.96). Most multilocus genotypes that were found in barley and wild barley were confined to their respective hosts except MLG-2, which was predominant on wheat but also infected barley and its wild relatives. MLG-2/MLG34 was also responsible for 2023 epidemic in all wheat-growing regions of Uzbekistan and showed temporal maintenance during the 2024 cropping year. This lineage may have resulted from hybridization between P. striiformis f. sp. tritici and P. striiformis f. sp. hordei although the source of parental lineages is still unknown, which requires further investigation. Monitoring the migration patterns of this lineage and estimating its pathogenic impact on overcoming different resistant genes is essential as it has the potential to cause epidemics in both wheat and barley.

The Toll/interleukin-1 receptor (TIR) domains are widely conserved across prokaryotes and eukaryotes. In plants, TIR nucleotide-binding leucine-rich repeat (TNL) proteins serve as the intracellular receptors that defend against pathogens. However, monocots lack TNLs. Wheat (Triticum aestivum) has few TIR domain-containing proteins, and their biological functions remain uncharacterized. Here, we report the identification of a TIR-nucleotide-binding site (NBS)-tetratricopeptide repeat (TNP) protein, TaTNP1, that is involved in wheat resistance against Puccinia striiformis f. sp. tritici (Pst). The P-loop motif in the NBS domain is required for TaTNP1 overexpression-conferred resistance. By contrast, TaTNP1-knockout lines exhibited compromised PAMP-triggered immunity and enhanced susceptibility to Pst. These genetic data indicate that TaTNP1 functions as a positive regulator of wheat immunity. Furthermore, this study demonstrated the contribution of wheat Enhanced Disease Susceptibility 1 (TaEDS1) to Pst resistance using TaEDS1-silenced and-overexpressing lines. In contrast to the TIR-only protein TaTIR, which is an active TIR that triggers TaEDS1-dependent cell death, TaTNP1 plays a non-catalytic, stabilizing role in immunity by directly binding to TaEDS1 and protecting it from proteasomal degradation. Collectively, our findings reveal new insights into how TIR domain-containing proteins modulate wheat immunity, not only through catalytic production of signaling molecules but also via direct binding and stabilization of TaEDS1.

Fusarium head blight (FHB) is a devastating fungal disease in wheat, causing significant yield losses and deterioration of grain quality under severe conditions. In this study, a genome-wide association study was conducted with 448 accessions using genotyping data generated by the 660K SNP array. Nine relatively stable FHB resistance loci were identified on chromosomes 1B, 1D, 2D, 5B, 7A, and 7B. Each QTL accounted for 4.1 to 10.4% of the phenotypic variation. Among them, QFhb.nwafu-7BS and QFhb.nwafu-7BL are novel loci. Polymorphisms of the flanking AQP marker AX-94527414 were developed for QFhb.nwafu-7BL, which could be useful for marker-assisted selection of FHB resistance. Correlation analysis indicated that variation in FHB response was independent of plant height, spike length, and uppermost internode length across the three environments. These results offer new resistance resources for FHB resistance breeding and insights for marker-assisted selection and gene cloning.

Apoplastic acidification represents a pivotal mechanism in the co-evolutionary dynamics between plants and pathogens. However, the mechanisms underlying this process remain largely uncharacterized. In this study, we unveil a mechanism by which the stripe rust fungal (Puccinia striiformis f. sp. tritici; Pst) effector manipulates plasma membrane (PM) H+-ATPases to promote apoplastic acidification and attenuate host immune responses. We identified a wheat (Triticum aestivum) PM H+-ATPase (TaHA2) as a key regulator of apoplastic pH and defense responses to Pst infection. The overexpression of TaHA2 exacerbated apoplastic acidification and Pst susceptibility, whereas the CRISPR-Cas9-mediated inactivation of TaHA2 in wheat conferred broad-spectrum resistance against multiple rust pathogens without compromising agronomic traits. Mechanistically, we found that the wheat calcineurin B-like interacting protein kinase 9 (TaCIPK9) phosphorylates TaHA2 at Ser-933, triggering intramolecular interactions between its C-terminal autoinhibitory domain and the central loop, thereby suppressing TaHA2 activity. Conversely, the CFEM (common in fungal extracellular membrane)-containing Pst effector PstCFEM2 competitively binds to the C-terminus of TaHA2, disrupting TaCIPK9-mediated phosphorylation and relieving autoinhibition. This effector-driven activation of TaHA2 amplifies apoplastic acidification and stomatal opening, ultimately dampening plant immunity. Our findings reveal a mechanism by which pathogens promote infection by subverting host pH regulation and provide a theoretical framework for engineering disease resistance through the manipulation of susceptibility genes.

The plasticity of crop development is crucial for survival and yield stability under adverse conditions. Saline-alkaline soil is a major environmental constraint limiting wheat productivity. Elucidating the regulatory basis of wheat developmental plasticity under salt stress is crucial for improving salt tolerance and yield stability. In this study, salt stress promotes the initiation of lateral root (LR) primordia while inhibiting LR emergence in wheat. Upon return to non-stress conditions, these primordia rapidly develop into LRs, enabling swift recovery and root system expansion. We identify glycogen synthase kinase 3 (TaGSK3) as a molecular switch that regulates this plastic response via brassinosteroid and auxin signaling pathways. By this mechanism, environmental signals are transduced into root development plasticity via TaGSK3 phosphorylation. This work provides new insights into how crops control developmental plasticity under stress.

Wheat stripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a destructive disease affecting wheat production because of its capacity for long-distance dispersal. This pathogen exhibits high genetic diversity, yet the low resolving power of currently used molecular markers has restricted in-depth understanding of its migration routes. We previously detected variations among isolates in the ribosomal intergenic spacer 1 (IGS1) region of Pst. In the current study, Pst IGS1 fragments were amplified directly from DNA extracted from wheat rust lesions each bearing a single uredinium, and the intraspecific variation pattern of IGS1 was comprehensively analyzed. Thirty polymorphic loci characterized by variations in repeat units and subunits were detected, with up to 12 polymorphic loci identified within single isolates. Among the 3,050 field samples examined, 506 distinct haplotypes were identified based on combinations of these polymorphic loci, demonstrating that the IGS1 haplotypes can be effective markers for revealing genetic diversity of Pst. Furthermore, IGS1 haplotypes allowed dissection of genetic structure and gene flow among different Pst populations. The IGS1 haplotype genotyping system features several advantages: it requires no fungal isolation or culturing, relies on only one pair of specific primers, and supports high-throughput sample processing. It therefore represents a powerful tool for elucidating the gene flow and genotype flow among populations, as well as for tracking long-distance dispersal of Pst both on regional and continental scales.

Sustaining wheat yield gains requires optimizing the spatiotemporal coordination of source (leaves), transport (stems), and sink (spikes) organs. However, the physiological mechanisms and underlying genetic networks orchestrating the dynamic development of these critical structures remain largely uncharacterized. Here, we leveraged high-resolution time-series phenotyping across 590 wheat accessions evaluated across three year-site environments (comprising two locations and two growing seasons) to dissect the genetic architecture of these biomass partitioning trajectories. To fully capture this spatiotemporal regulation, our analysis explicitly integrated both the temporal tracking across five floret developmental stages (Z39-Z65) and the spatial partitioning among these organ-specific dynamic systems. We identified 36 multi-stage stable dynamic quantitative trait loci (QTL) regulating five source–sink-related traits. By constructing genetic association and epistatic interaction networks, we prioritized two pivotal dynamic QTL, namely Qa.nw-7B.848 and Qa.nw-1D.96. Multi-omics integration pinpointed TraesCS7B03G1340600 as a key candidate gene for Qa.nw-7B.848. Furthermore, haplotype analysis uncovered distinct selection footprints, demonstrating how specific allelic combinations have been differentially selected to optimize yield components across diverse geographical environments. Collectively, this study moves beyond static trait analysis, offering a dynamic genetic framework and specific epistatic targets to precision-design wheat architecture for enhanced productivity.

The emergence of triadimefon resistance in Puccinia striiformis f. sp. tritici (Pst), the causal agent of wheat stripe rust, threatens global food security. Although the novel SDHI fungicide flubeneteram shows high efficacy, the molecular basis of resistance remains unclear. Here, we identify the ABC transporter gene PstABCG2 as a key multidrug resistance (MDR) determinant in Pst. Transcriptomics revealed its sustained upregulation under fungicide stress. Silencing PstABCG2 via RNAi and HIGS enhanced fungal sensitivity to both fungicides, while heterologous expression in Fusarium graminearum restored resistance. Structural and biophysical analyses identified E1184 as a critical binding residue-its mutation (E1184Y) impaired triadimefon affinity but preserved flubeneteram binding via adaptive halogen bonding. Furthermore, the GATA-family transcription factor PstGATA directly activated PstABCG2 expression, and its silencing phenocopied the hypersensitivity of PstABCG2-silenced isolates. The results provide a theoretical foundation for elucidating multidrug resistance mechanisms in Pst, field resistance management, and novel fungicide target development.

Puccinia striiformis f. sp. tritici (Pst) is a destructive pathogen renowned for its dual reproductive modes, an asexual stage on wheat and a sexual stage on barberry (Berberis), which makes wheat cultivars vulnerable to newly emerging races. However, little has been known about the impact of treatment on barberry plants on declining population genetic diversity and race composition. In this study, we selected six barberry-wheat coexisting sites in which Pst occurs sexually as treatment and control groups for experimental purposes. The treatment group was treated with triadimefon fungicides on barberry at the early pycnial stage to suppress sexual reproduction. Conversely, the control group represented sexual reproduction without fungicide application. Pst populations from wheat fields close to barberry with and without fungicide treatment were phenotyped on Chinese differentials and genotyped using a DNA microarray. The results showed the treatment population displayed a lower heterozygosity level (Fhom = -0.36945, Tajima's D = 0.285033) and a lower genetic diversity (π = 0.000156053) compared with the control population (Fhom = -0.41745, Tajima's D = 0.955451, π = 0.000184483), and a significant difference was observed between both populations (P < 0.001; P < 0.05). The treatment population identified 17 new races and 3 known races, and the control population identified 46 new races and 8 known races. The treatment population (α = 4.644) showed a lower diversity of the standard races than that of the control population (α = 5.194) based on a set of 25 Yr-single gene lines. Our results showed the significant impact of fungicide application on barberry to minimize the emergence of new races and the level of genetic diversity. This study provides guidance to growers, emphasizing the importance of timely fungicide application on barberry to ensure the long-term resistance durability of wheat cultivars against stripe rust.

Endoplasmic reticulum (ER) stress and the hypersensitive response (HR) are recognized as cornerstones of plant immunity; however, the mechanistic synergy and the strategies pathogens employ to dismantle this alliance remain elusive. Here, we identify a virulence effector, PstCRT (calreticulin), from Puccinia striiformis f. sp. tritici (Pst), that suppresses host immune responses by disrupting ER stress-mediated HR. PstCRT directly targets the HR-like lesion-inducing protein (TaHRLI) in wheat and obstructs its ER translocation. Within the ER lumen, TaHRLI interacts with wheat calreticulin (TaCRT), triggering Ca²⁺ efflux and activating the unfolded protein response (UPR) to induce cell death and disease resistance. Pathogen-derived PstCRT structurally mimics TaCRT to sequester TaHRLI to the plasma membrane via competitive interaction, thereby effectively suppressing ER stress-induced HR initiation. Crucially, we found that CRT secretion represents a conserved virulence strategy across different rust genera. AlphaFold-guided engineering of TaHRLI in wheat generated the TaHRLIMut variant that evades PstCRT recognition. Overexpressing TaHRLIMut in wheat conferred broad-spectrum resistance against Pst in biennial field trials, effectively mitigating pathogen-induced yield losses while preserving essential agronomic traits. Collectively, this study elucidates a molecular mechanism underlying pathogen disruption of ER stress-induced HR to promote infection and proposes an innovative strategy for engineering durable crop protection.

Crown rot (CR), predominantly caused by Fusarium pseudograminearum (Fp), is a significant disease of wheat worldwide. However, existing CR inoculation methods and resistance assessment systems lack efficiency, consistency, and precision. Here, we developed a hydroponic inoculation method for wheat seedlings and an integrated CR resistance assessment system that combines disease index (DI) and in planta fungal biomass. The results show that hydroponic inoculation at 24°C with a conidial concentration of 5 × 106 conidia/ml reduces the experimental duration from 21-45 to 18 days. For the integrated system, we refined CR symptom evaluation criteria for DI calculation and incorporated fungal biomass quantification (fungal DNA/wheat DNA), ensuring accuracy and reliability. Verification using five wheat varieties confirmed the system's precision. Sunco exhibited near resistance (DI = 25.71; biomass = 0.41), and Mingxian169 showed moderate resistance (DI = 39.84; biomass = 0.44), while Xiaoyan22, Fielder, and Xinong979 displayed high susceptibility (DI > 60, biomass > 0.63), consistent with previous studies. Our methods will facilitate large-scale germplasm screening and advance research on F. pseudograminearum-wheat interactions.

Stripe or yellow rust (YR), caused by Puccinia striiformis Westend. f. sp. tritici (Pst), is a devastating fungal disease of wheat. Here, we report the map-based cloning of Yr26 (synonyms: Yr24, YrCH42, and YrG22), a major stripe rust resistance gene, and its validation by gene silencing, mutation analysis and transgenic complementation. Yr26, which firstly arises in emmer wheat and recently has introgressed into bread wheat, is a newly evolved gene encoding a transmembrane protein. AlphaFold v3.0 predictes a C-terminal four-helical bundle domain that is critical for triggering Ca2+-dependent cell death, a hallmark of the hypersensitive response. The cloning of Yr26 offers not only a genetic resource for pyramiding stripe rust resistance genes in wheat breeding but also an opportunity to study host–pathogen co-evolution. Yellow rust (YR) is a devastating fungal disease of wheat. Here, the authors report the cloning of Yr26 as a transmembrane protein and reveal its evolves within the Triticeae lineage as an orphan gene.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a major constraint to wheat production in China. Although the disease occurs in Tibet every year under the favorable weather conditions, the virulence diversity and population structure of the Pst population in this region remain poorly understood. In this study, 312 Pst isolates collected in 2025 from five major wheat-growing regions of Tibet (Qamdo, Nyingchi, Lhasa, Shannan, and Shigatse) were characterized using virulence phenotyping and the Pst 5K genotyping-by-target-sequencing (GBTS) platform. Virulence testing on 19 Chinese differentials identified 139 races, including 88 previously reported and 51 novel races, with Su11-3, HY-009-1, and CYR34 being the predominant races. Testing the isolates with 18 Yr single-gene differentials identified 190 virulence phenotypes. None of the isolates were virulent to Yr5 or Yr15, whereas virulence frequencies to the remaining Yr genes ranged from 2.2% to 89.7%. Population genetic analyses using the GBTS data revealed clear regional differentiations. The Nyingchi population formed a distinct cluster with relatively low genomic diversity despite maintaining considerable virulence diversity, whereas the Lhasa and Shannan populations showed extensive admixture.The Qamdo and Shigatse populations consisted of unique genetic components and exhibited relatively high levels of virulence and genetic diversities. Combined virulence and genomic analyses indicated region-specific epidemiological characteristics and suggested that Qamdo may represent an important center of Pst diversification, whereas Lhasa and Shannan may facilitate pathogen dispersal and gene exchange. This study provides the first comprehensive assessment of Pst populations across Tibet and offers valuable information for disease surveillance, resistance breeding, and integrated management of wheat stripe rust in Tibet and neighboring wheat-growing regions.