Morphological and genetic differences of three Sinibrama macrops populations from Qiandaohu Lake were compared by traditional morphological method and mitochondrial D-loop region sequence analysis. Based on the results of principal components analysis, specimens clustered together by scatter plots of scores could not be divided. It indicated that there is no morphological differentiation among all samples from Qiandaohu Lake. 27 polymorphic sites were detected yielding 72 haplotypes. NJ tree and network showed that the geographic distribution was ambiguous. There were similar base composition among the three populations, and the content of A T (64.82%) was higher than the content of G C (35.18%). High genetic variation (h) and low nucleotide diversity (π) were found in the three populations analyzed for D-loop, and the diversity of Fenkou (h=0.968?0.018, π=0.00476?0.00049) was the highest, Linqi (h=0.968?0.017, π=0.00453?0.00041) was the second, Fuwen (h=0.937?0.037, π=0.00330?0.00035) was the lowest. The values of genetic differentiation index (Fst) were lower than 0.05 and not significant (P>0.05). A strong gene flow among the populations were observed (Nm>4), and the Nm between Fenkou and Fuwen was highest (Nm= 27.40). The results of all supported the three geographical populations belong to a randomly mating population. Consequently, we suggest to keep the pattern of gene flow of S. macrops, and reduce the influence of human activities so as to protect this important species in fishery resources.