A total of 5 556 microsatellite sequences were found from ESTs databases (45 318 unigenes) which were constructed from brain,muscle,and liver tissues of Grass Carp(Ctenopharyngodon idella).According to these microsatellite sequences,118 pairs of primers were designed with Primer Premier 5.0.Nineteen pairs of microsatellite primers could be used to successfully amplify clear and highly polymorphic products by method of PCR.These EST-SSRs primers were employed to detect the genetic diversity of three groups of Grass Carp populations from the Yangtze River System (Shishou,Jianli,Changsha) and two groups from the Pearl River System (Qingyuan,Zhaoqing).A total of 93 polymorphic loci were amplified and 2 to 8 alleles or 4.89 alleles on average were detected by each primer.The average polymorphism information content (PIC) ranged from 0.415 4 to 0.460 4,indicating that Grass Carp population had lower genetic diversity; the average observed heterozygosity (Ho) was from 0.415 8 to 0.501 3 and the average expected heterozygosity (He) was from 0.450 6 to 0.5028.Changsha group had the highest mean expected heterozygosity (0.502 8),while Jianli group had the lowest mean expected heterozygosity (0.450 6) and mean observed heterozygosity (0.415 8).These observations indicate that Changsha group has the highest genetic diversity,whereas Jianli group has the lowest; F_st value indicates that the populations are lowly differentiated.After applying Hardy-Weinberg equilibrium (HWE) test,several loci were found to be significantly deviated from HWE in five populations.The dendrogram based on genetic distance(D) showed two major clusters according to basin distribution of Grass Carp populations: the stocks from Changsha,Shishou and Jianli,which were sampled from the Yangtze River,were in one cluster; while the other cluster consisted of Zhaoqing and Qingyuan populations sampled from the Pearl River.