Abstract: To investigate population differentiation of Cultrichthys erythropterus, 186 samples were collected from 6 localities in Poyang Lake (HK, XZ, DC, PY, YG) and Dongting Lake (DTH) to analyze the morphology and Cyt b sequence. The results showed that there were no significantly morphological differences among populations. The genetic differentiation coefficient (Fst) ranged from -0.00567 to 0.33429, indicating evidently genetic differences among populations that further supported by the analysis of haplotype distributions and ANOMA. The NJ tree of the six populations revealed that the XZ, DC, PY and YG population had the closest relationship that was clustered to form one branch, and DTH population was clustered to all other populations for another branch except HK that is the transitional population between these two branches. Perhaps, C. erythropterus was the lacustrine fishes, which resulted in long-term isolated history and limited gene flow.