Ten microsatellite loci was used to investigate the genetic variation and differentiation of complete set line breeding populations (parental lines A and B of selective F₄ and their reciprocal crosses AB, BA) and wild population (YZ) sampled from the Yangtze River Estuary. The results indicated that: (1) Genetic diversigenetic diversity of the four breeding populations was lower than that of the wild populations. (2) The results of molecular variance analysis (AMOVA), NJ phylogentic tree based on Nei’s genetic distance, principal components analysis (PCA) and STRUCTURE genetic cluster analysis consistently showed that there were significant differentiation (<0.01) among three clusters, namely, AB and AA clustered one group, BA and BB clustered another group and YZ consisted of separate group. (3) According to selective pressure analysis in the Arlequin software and Bayescan software, three microsatellite loci in the breeding populations and one locus in the wild population were detected with significantly positive selection pressure, indicating the continuous strong artificial selection would have resulted in a high level of genetic differentiation between breeding and wild populations. In general, the artificial selection has decreased the genetic diversity within breeding populations and enlarged genetic differentiation between breeding and wild populations. Fortunately, the complete set line breeding could enrich genetic diversity of breeding populations.