Abstract: This study was conducted to evaluate the genetic structure of B. xanthopterus populations in Karoon, Krkhe and Jrahi rivers in southwest of Iran using Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA). Amplified mtDNA (cytochrome b and part of tRNAGlu) genes were digested with nine restriction enzymes in order to check the presence of recognition sites. RFLP analysis revealed a total of nine composite haplotypes in 100 individuals. The estimated sequence divergence between all pairs of haplotypes ranged from 0.8 to 4.4%. The haplotype and nucleotide diversity within populations ranged from 0.3785 to 0.6456 and 0.0052 to 0.0125, respectively. The average nucleotide diversity (πxy) and the net nucleotide divergence (δ) among 3 populations ranged from 0.0108 to 0.0127 and 0.00 to 0.39%, respectively. The neighbour-joining dendrogram which was constructed based on the net nucleotide sequence divergence showed that B. xanthopterus populations separated in two groups. The B. xanthopterus populations in Karoon and Krkhe rivers are categorized in one group and Jrahi river in a second group. The obtained phylogenetic relationships was approved by Fitch-Margoliash method using Slatkin linearized FSTs. The obtained results at the present study showed a low genetic diversity between B. xanthopterus of Karoon, Krkhe and Jrahi rivers in southwest of Iran. The results also suggested that for maintaining of genetic diversity of B. xanthopterus at appropriate levels, the exchange and transfer of Barbus stocking between Jrahi and other river basin should be avoided.