Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, with a prevalence of 1 in ca. 1000 individuals and accounting for 10% of the European dialysis population. It is most frequently (75-80%) caused by mutations in the PKD1 gene encoding Polycystin-1. Besides systemic manifestations such as liver cysts, hypertension and cardiac abnormalities, the most prominent phenotype is the gradual development of kidney cysts, leading to kidney enlargement and a decline in kidney function leading to end-stage kidney disease at an average age of ca. 50 years. The cysts arise from tubular epithelial cells in the nephron that start to dedifferentiate, proliferate and eventually secrete fluid into the cyst lumen promoting its swelling. This process is molecularly regulated by changes in Ca2+, cAMP, PKA and PI3K-Akt-mTOR signaling, among others. The renal cysts are further characterized by ciliary dysfunction, altered signal transduction, loss of polarity, apoptosis, mitochondrial abnormalities, metabolic reprogramming, inflammation, oxidative stress and epigenetic changes. However, the exact sequence of events and their relative importance in the process of cyst formation remains unclear. Therefore, we analyzed the transcriptomic cyst hetero- and homogeneity to identify the pathways that were different, but also shared among individual cysts. Method We performed RNA-Seq analysis on 40 individual cystic membranes of nephrectomized kidneys of 4 PKD1 patients. We included 4 samples of macroscopically healthy tissue from 2 ADPKD kidneys (histologically confirmed to have microcysts) and 4 samples from 4 healthy kidneys. Principal component, cluster and trajectory analysis was performed to explore cyst heterogeneity. Adjacent pieces of cystic membrane were analyzed for histology (H&E, Sirius Red) or stained with FITC-tagged Lotus Tetragonolobus Lectin (LTL) or Dolichos Biflorus Agglutinin (DBA), a proximal and distal tubular marker, respectively. Results Although the cysts clearly separated from the healthy tissues in the analyses, there was also a distinct heterogeneity observed among the cysts that was independent of patient, gender, cyst size, location or fluid color. Pathway analysis revealed that these changes were characterized by a gradual increase of interstitial (remodeling) and inflammation markers, and a gradual decrease in differentiation markers (e.g. development, intercellular junctions, basement membrane) along the trajectory pseudotime. We could identify 6 clusters of cystic samples, with 2 clusters deviating from the main trajectory. With respect to tubular/epithelial dedifferentiation, the decrease in proximal tubular markers already occurred in microcystic tissue and was stronger compared to the other tubular segments. Consequently, none of the analyzed cystic membranes stained positive for LTL, while others showed clear staining for DBA. Microcysts on the other hand stained positive for LTL or DBA. Several cysts showed RNA marker expression of multiple tubular segments. Indeed, smaller macrocysts (diameter in the mm range) were lined by both LTL- and DBA-positive regions. Conclusion By analyzing transcriptomic hetero- and homogeneity among individual cysts, the molecular alterations in ADPKD progression can be tracked down and determined whether they occur similarly in all cysts or at different levels. Our data also suggest that loss of proximal tubular markers in macrocysts is more prominent compared to the decrease in distal markers. Finally, inner cyst walls can consist of cyst-lining cells of multiple origins.