Shirbot (barbus grypus) belongs to Cyprinidae family and it is considered as one of the most important commercial species in Iranian aquaculture. It has a large presence in the west and southwest water resources of Iran specially Khuzestan Province rivers. However, there is lack of cellular information about this species. In this study, the genetic structure of barbus grypus from Dez and Karoun regions was investigated by 7 polymorphic microsatellite loci. According to the results, the Fst value was 0.025 which indicates the low genetic differentiation between the populations. In Hardy-Weinberg equilibrium investigation, most of the loci showed deviation from equilibrium. The number of alleles, observed and expected heterozygosity were, 7-20, 0.16-1(the average: 0.7) and 0.736-0.933(the average: 0.856), respectively. This indicates the allelic diversity and genetic variation of investigated populations is at favourable level. Also, analysis of molecular variance showed there is low genetic variation among populations and most of the observed variation is within the populations. Results derived from genetic distance indicated that Shirbot populations are separate from each other in the investigated regions.