Persian squirrel (Sciurus anomalus) is the significant mammal of Zagros Oak Forest in West of Iran, but our knowledge about such valuable animal still is little. In this study inter-simple sequence repeats (ISSR) markers were applied to assess genetic diversity and genetic relationships of 42 Persian squirrels caught from three different regions (region 1: Tajan, Region2: Vargool, region3: Goshkhani) in Kurdistan province, Iran. Genomic DNA extracted from the hair follicles of Persian squirrels. Six di-nucleotide repeat primers used to detect the level of polymorphism. Three out of six used preliminarily primers produced clear bands and used for polymorphism detection. From 56 generated bands, 89.28% were polymorphic. The genetic diversity (mean ± SD) for the regions 1 to 3 was: 0.334 ± 0.1660; 0.262 ± 0.2148; 0.2544 ± 0.2133 respectively. Genetic distances based on Nei’s unbiased method revealed a notable similarity between populations 2 and 3 while populations 1 and 3 were substantially different. These results were compatible with the population’s geographical distances.