To know the genetic variation and relationship of Xenocypris davidi populations between Qiandao Lake and Yangtze River, we analyzed mitochondrial COⅠ sequences of Xenocypris davidi of five geographic populations at Qiandao Lake (Fenkou County, Fuwen County and Linqi County of Zhejiang Province) and the other areas (Jingxian County of Anhui Province and Nanchang County of Jiangxi Province) to detect their genetic structure and genetic variation. 10 variation sites were observed and 10 haplotypes were defined in 92 samples sequences. In the network of sequences haplotypes of Xenocypris davidi,the distribution pattern of haplotypes presented a certain degree of corresponding relationship to geographic pattern. Analysis of fixation index (F_st) showed that there were obvious variation between Yangtze River and Qiantang River(0.110 55-0.453 67), and no variations was detected in populations of Qiandao Lake(F_st≤0.066 05), and the variation between Yangtze River's Jingxian and Nanchang was very high(F_st=0.399 93) .Genetic distance between each two populations indicated that three populations in Qiandao Lake had close relationship,however, Jingxian County displayed highest variation with others. Both Neutrality test and Mismatch distributions of all populations indicated that population outbreak and population expansion may have occurred in Nanchang population in history, while other populations showed no obvious population expansion.