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The application of microsatellite simple sequence repeat (SSR) markers has proven to be highly effective in genetically characterising the origins of various fruit species. Bosnia and Herzegovina boast abundant cornelian cherry (Cornus mas L.) germplasm within its natural habitats. This study focuses on three geographically distant regions in Bosnia and Herzegovina–Drvar, Mostar and Zenica–comprising a sample of 60 cornelian cherry genotypes. The genetic analysis of cornelian cherry utilised eight microsatellite primers to characterise the genetic makeup of the 60 genotypes. Notably, all observed loci in the three populations demonstrated 100% polymorphism. The total genetic differentiation is 0.042, which is also confirmed by the analysis of molecular variance (AMOVA), which indicates that 3% of the total genetic variation is between groups, 8% between individuals and 89% within individuals. The results of pairwise Fst analysis (pFst) indicate that the greatest differentiation is between the populations of Mostar and Zenica (0.037), between Drvar and Zenica (0.026) and between Drvar and Mostar (0.025). The results of the genetic distance according to Nei confirm these results. Also, the largest difference was observed between the populations of Mostar and Zenica (0.316), then between Drvar and Zenica (0.285), and the smallest between Drvar and Mostar (0.251). This research contributes valuable insights into the genetic diversity and characterisation of cornelian cherry populations in different regions of Bosnia and Herzegovina.