Myotis myotis (Borkhausen, 1797) and Myotis blythii (Tomes, 1857) are genetically close species of bats with very similar morphology and karyology. In this study, specific descriptive markers of 3 species ( Myotis myotis, Myotis blythii, and M. cappacinii ) belonging to the genus Myotis were determined by using ISSR technique for the first time. In total, 91 bands including 86 polymorphic bands were obtained from the 7 best-optimized primers out of the analyzed 16 primers. UPMGA cluster analysis showed 4 separate clusters. According to these results, the M. myotis and M. blythii were divided into two close groups, whereas a single individual belonging to M. myotis colony formed the third group. M. cappacinii was in a more distinct and remote group with respect to sibling species. Genetic diversity for all populations was calculated as 94.5%. ISSR-PCR method was shown to be a reliable and useful technique for detecting genotypic similarities/differences of Myotis species.