Generally, the lifespan of a Chinese mitten crab (Eriocheir sinensis) is two years, and then the crab would be died after it finishes the reproduction. Theoretically, the Chinese mitten crabs of adjacent two years (e.g. 2010 and 2011) would not conduct genetic mating or gene flow. There is a highly hypothesis that there were two lineages or categories of the odd and even years in this crab. In this paper, genetic differentiation was analyzed and the hypothesis was tested using seven wild populations (sampled in 2004、2006 ~ 2011) from the Yangtze River based on 18 microsatellite loci. The results indicated that: ① the HO and HE of the odd year populations were significantly lower than these of the even year populations (P < 0.05 ); ② There were significant pairwise FST difference (P = 0.024) between the odd and even year populations; ③The phylogenetic dendrograms based on NJ methods showed obvious two clades, one clade consisted of the even year populations, another consisted of the odd populations; ④ Both Principal coordinate analysis and STRUCTURE genetic clustering analysis indicated that significant genetic differentiation between the odd and even year populations, but slight genetic introgression would had occurred between them; ⑤ Genetic bottleneck signal was detected in the two even year populations (2004, 2006) and odd year populations (2007, 2011). The results of this study demonstrated that the Chinese mitten crab in Yangtze River exist two lineages of the odd and even year populations, however, there were a level of gene flow between the two lineages.