Abstract: In this study, we selected 181 microsatellite loci from 2400 EST sequences to perform germplasm identification and genetic diversity analysis in different regions of grass carp, Ctenopharyngodon idella. Nine pairs of primers, which could give stable and polymorphic amplification profiles, were screened out from 46 microsatellite loci and used to analyze the genetic structure of C. idella. The 9 pairs of primers were used to analyze the genetic structure of 8 populations of the Yangtze River (CSC, AQC, JXC, JJC, SSC, SJC, RCC and HJC) and 1 red population (HC). The mean value of number on alleles (Na) of CSC, AQC, JXC, JJC, SSC, SJC, RCC, HJC and HC was 4.67, 5.22, 5.33, 5.00, 4.89, 4.78, 4.89, 4.67 and 2.56, respectively. The mean value of expected heterozygosity (He) was 0.6397, 0.6543, 0.6831, 0.6356, 0.6737, 0.6483, 0.6664, 0.7129 and 0.4696, respectively. And the average value of polymorphism information content (PIC) was 0.5787, 0.6126, 0.6283, 0.5894, 0.6217, 0.5956, 0.6136, 0.6582 and 0.3949, respectively. It demonstrated that the genetic diversity of HJC was the highest and HC was the lowest among the nine populations. Using unweighted pair-group method with arithmetic means method (UPGMA) based on their genetic distances, the cluster analysis in nine populations showed that 8 populations of the Yangtze River first grouped together, then they clustered with the red population. In addition, the genetic distance between AQC and SJC was 0.0725, which was the nearest, the genetic distance between HC and CSC was 0.5217, which was the farthest. Our results will have important value in germplasm resources preservation, germplasm identification and breeding of C. idella.