Shirbot (barbus grypus) belongs to the Cyprinidae family and is widely present in the west and Southwest’s water resources of Iran, especially the Karkhe River in Khuzestan Province. The aim of this study was to compare levels of genetic polymorphism between Karkhe River and cultured Barbus grypus populations using seven microsatellite loci. Genetic diversity was investigated by studying 60 samples collected from two regions.  According to the results, the F_st value was 0.033, which indicates low genetic diversity between the populations. Most of the loci showed deviation from Hardy-Weinberg equilibrium. Also a relatively high level of gene flow was found among the population. Genetic variations in Karkheh: mean number of alleles per locus, N_a=10.286, mean effective number of alleles, N_e=6.789, observed heterozygosity, H_o=0.691 and expected heterozygosity, H_e=0.840 and cultured fish N_a=13.286, N_e=9.141, H_o=0.800 and H_e=0.883 were not statistically different. Also, analysis of molecular variance showed that there is low genetic variation among populations and most of the observed variation is within the populations.