To determine genetic diversity of Paracobitis hircanica in Tilabad, Touskestan and Zaringol rivers of Golestan province, six microsatellite markers were utilized all of which showed polymorphism. Investigation of genetic differentiation revealed the Fst of 0.028, indicating low genetic differentiation among the samples of the studied regions. Also, investigation of Hardy-Weinberg equilibrium showed significant deviation from Hardy-Weinberg equilibrium in 16 samples of 18 tests of population-loci mostly due to the observed heterozygosity deficiency. The number of alleles and observed heterozygosity was 8-16 (the average of 11.94) and 0.03-1/00 (the average of 0.52), respectively, which are slightly higher than the amount reported for fresh water fish. Results indicated that P. hircanica has high allelic richness and considerable genetic diversity in the investigated regions. Additionally, analysis of molecular variance revealed that most of the observed diversity was within populations and there is low diversity among the populations. Results from genetic distance represented that there is probably separate populations of P. hircanica in Golestan Rivers