The genetic population structure of Capoeta aculeata from the Zagros Basin including three rivers (Beshar, Khersan, and Maroun) was investigated using the partial mitochondrial DNA (mtDNA) control region (D-loop) sequences (n=۹۰) and eight microsatellite markers (n = ۱۲۰). To address this issue, genetic structure differences between the three populations were evaluated by haplotype diversity and pairwise comparison based on allelic distribution. Both methods used were concordant in that they showed low to moderate levels of genetic variation and suggested that C. aculeata populations were partly diversified. For the mtDNA method, the average haplotype diversity (h) and nucleotide diversity (π) were ۰.۸۴۶±۰.۰۲۷ and ۰.۰۰۳۷±۰.۰۰۵, respectively. For the microsatellite markers approach, the average number of alleles per locus ranged from ۴ to ۱۴, while the average observed heterozygosity (Ho) at various loci varied between ۰.۲۱۲ and ۰.۵۷۹. The Hardy-Weinberg test showed that the microsatellite loci deviated significantly in the populations indicating a deficit of heterozygotes. Besides, the results of pairwise FST estimated and analyzed molecular variance (AMOVA) alongside neighbor-joining and structure analyses showed that most of the variations occurred between samples and finally significant genetic differentiation between populations for both mtDNA and microsatellite markers. At least two genetically distinct management units were observed using the two methods among the sampling sites. The non-significant differentiation between C. aculeata samples from the Khersan and Beshar rivers can be explained by a relatively recent disconnection of these two populations and/or small amounts of contemporary gene flow between the two gene pools. The analyses of both mtDNA and microsatellite markers provided potential markers for identifying probable populations and characterizing the conservation genetics of C. aculeata.