In this study population genetic structure of freshwater crayfish (Astacus leptodactylus) in Iran was investigated using direct sequencing of mtDNA control region. A total of 132 samples were collected from the different locations. The quality and quantity of total DNA were determined by agarose gel electrophoresis ethidium bromide staining and spectrophotometery, respectively. The results showed that 38 haplotypes were observed between samples in sequencing analyses. The haplotype diversity (h) and nucleotide diversity (π) were 0.811±0.049 and 0.0127±0.0038 sequencing techniques, respectively. Tajima’s D and chi-square (χ²⁾ values were 3.120 and 78.25, respectively. The significant positive D value for the samples might suggest balancing selection. Significantly negative Fu's Fs values and significantly positive Harpending test values were taken as evidence of a population expansion.The results of F_ST ,Exact test and analysis of molecular variance (AMOVA) demonstrated that samples between Siahdarvishan River , Jafrood River and Astara region in the southwest Caspian sea statistically are significant in sequencing techniques (P<0.0001).Therefore three distinct groups were identified.  These results showed that haplotype distributions in different locations were significant and populations of Siahdarvishan River and Astara region statistically were significant (P < 0.0001). These results suggests that the unique genetic structure of Siahdarvishan River, Jafrood River and Astara region represent a highly valuable genetic resource and provide useful information for identifying populations and genetic improvemnet.