The Chinese mitten crab Eriocheir sinensis is one of the most economically important aquaculture species in China due to its taste and nutritional value. In this study, the genetic status of both wild and cultivated stocks of E. sinensis was assessed using simple sequence repeat (SSR) markers. Ten microsatellite markers reported in previous studies were selected to estimate the level of genetic diversity within one Haihe natural population and three selected artificial populations (Yangtze No.1, Guanghe No.1, and Qilihai crab) and to compare the degree of genetic differentiation among them. Unique PCR products and high levels of polymorphism were observed for all loci. For the four populations of E. sinensis, the number of alleles per locus ranged from 3 to 17, with a mean allele number of 8.5 to 9.7. The mean observed heterozygosities ranged from 0.566 to 0.661, while the mean expected heterozygosities ranged from 0.720 to 0.745. Additionally, the mean polymorphism information content ranged from 0.687 to 0.716. Furthermore, the inbreeding coefficient (Fis) ranged from 0.080 to 0.827, and there were 13 cases that significantly deviated from the Hardy-Weinberg equilibrium (P<0.05) among the 40 population-locus cases (4 populations × 10 loci). The results of the microsatellite survey indicated that the genetic diversity of the three artificially selected populations was slightly lower than that of the natural population, but still maintained at a high level for further breeding. The analysis of genetic differentiation showed that Fst ranged from 0.015 to 0.075, genetic similarity ranged from 0.7702 to 0.9401, and genetic distance ranged from 0.0617 to 0.2611. UPGMA phylogenetic analysis divided these populations into two groups. Natural population and Guanghe No.1 stocks were allocated to the same cluster, and the Qilihai crab was allocated to another cluster. In conclusion, the four populations of E. sinensis possessed high genetic diversity but low to moderate levels of genetic differentiation. The information on the genetic variation and differentiation obtained in this study will provide a theoretical basis for further breeding and utilization of germplasm resources of E. sinensis.