In this study, molecular characterization of four Prunus spinosapopulations was carried out using the inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) marker method. Populations were collected from Bolu, Düzce and Zonguldak provinces in Turkey. After genomic DNA (gDNA) isolation from the populations, PCR amplification was performed using 13 ISSR primers. As a result of the study, 116 polymorphic bands were obtained and the polymorphism rate was determined as 81.89%. These results were based on the genetic distance matrix obtained in the NTSYS-pc package version 2.10. The results showed that the closest genotypes were DY2 and DY4, with a similarity value of 0.2037 between them. DY4 and BM1 are found to have a similarity value of 0.5641, which indicates that they are the two genotypes that differ most from one another. The mean polymorphism information content (PIC) value was determined as 0.3507 and the mean Nei’s gene diversity (H) value was determined as 0.4559. In the unweighted pair group method with arithmetic mean (UPGMA) dendrogram, 13 populations were divided into two groups. According to principal component analysis (PCA), the first three eigen values explained 55% of the total variance in the population. As a result, the polymorphism rate was found to be very high with ISSR analysis, and the UPGMA dendrogram was compatible with the geographical distribution. The results of studies on P. spinosapopulations using the ISSR analysis can be beneficial for cultivar development and further studies in the future.