Genetic analysis using restriction fragment length polymorphisms (RFLPs) of cytochrome b in mtDNA was made to clarify genetic variations among two Iranian Rutilus rutilus caspicus populations of commercial importance from the southern coast of the Caspian Sea. Polymorphism was detected using six restriction enzymes and a total of six composite haplotypes were identified. Four haplotypes were rare occurring only once in two regions (west and east of the southern Caspian Sea). Nucleotide and haplotype diversities were higher in the south-west region of the Caspian Sea (π=3.43%, h=23.3).The nucleotide divergence between the two populations was low (0.064%). The test for heterogeneity of composite haplotype frequencies gave no significant outcome for all samples (χ2=0.137, P≤0.05). The results indicate that significant attention should be paid to the genetic characterization of R. rutilus caspicus populations for conservation of their genetic pools and aquaculture policies at the coastlines of the Caspian Sea.