The mtDNA cyt b gene of Odontobutis potamophila from five lakes was amplified and sequenced. Result showed that the average content of A+T(55.8%) was a little higher than that of G+C(44.2%),in the aligned sequences of 1141bp. 806 variable sites were detected and 87 haplotypes were recovered. The haplotypes diversity and nucleotide diversity were 0.969±0.012 and 0.20081±0.00742, respectively. The genetic distance between Lake Taihu population and Lake Dazonghu population displayed the lowest, up to 0.137, while that between Lake Chaohu population and Lake Dazonghu population was the highest to 0.424.The fixation indices of analysis of molecular variance among populations was 0.531, The phylogenetic tree of cyt b built with MEGA 4.1 showed that five populations were clustered into clades based on genetic distance. There was little general flow between O. potamophila populations from five lakes.