Opuntia ficus-indica Mill. commonly known as cactus pear is the most agronomically important species in Cactaceae. Turkey has important genetic resources of Opuntia spp. which should be characterized for further breeding strategies. In this study, molecular characterization of plant materials collected from different regions of Turkey in which Opuntia species grown naturally, was performed by using Random Amplified Polymorphic DNA (RAPD) markers. DNA was successfully amplified by 50 RAPD primers. Among 250 bands generated by the RAPD primers, 180 were polymorphic. The number of bands detected by a single primer set ranged from one to 12 (average of five bands/primer). The percentage of polymorphism was 72% based on RAPD data. All data were scored as discrete characters and unweighted pair group method with arithmetic mean (UPGMA) dendrogram and principle coordinate analysis (PCoA) were performed. Based on the results, Opuntia genotypes showed high genetic diversity and we showed that RAPD markers are powerful tool to discriminate Turkish Opuntia genotypes. The high genetic diversity existing in the Turkish germplasm suggests that it would be beneficial to utilize this pool in Opuntia breeding programs and germplasm management activities.