The Criollo Argentino horse is a national symbol and a heritage breed that represents both cultural identity and a key component of traditional production systems in Argentina. This study aimed to assess the genetic differentiation of the Criollo Argentino relative to other horse breeds and to infer potential factors shaping its diversity through the genomic analysis of 24 representative animals. Samples were genotyped using a medium-density SNP array containing 65,157 markers, and genetic diversity analyses were conducted across autosomal SNP sets filtered under different criteria. Contemporary effective population size was estimated using both linkage disequilibrium from SNP data (Ne = 155.6) and pedigree analysis (fe = 157.7), showing remarkable consistency between genomic and genealogical approaches. Measures of expected heterozygosity, Hardy–Weinberg equilibrium, and linkage disequilibrium showed that Criollo Argentino horses retain moderate to high levels of genetic variability. Results further revealed that the Criollo Argentino is a genetically well-defined population with high within-individual diversity and clear differentiation from other breeds. In addition, the Criollo Argentino population was compared with eight other horse breeds to evaluate genetic distance and population structure. Estimates of genetic distance, population structure, and fixation index values were consistent with the documented history and development of the breed, while PCA and Bayesian clustering analyses supported a shared origin between the Criollo Argentino and the Peruvian Paso, both derived from Iberian horses introduced to the Americas during colonization. Overall, this work provides genomic evidence that strengthens the historical and biological characterization of the Criollo Argentino and supports informed decision-making for breeding, management, genetic improvement, and conservation programs for the breed.
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