Microsatellites were firstly screened in the Siniperca chuatsi gonadal transcriptome obtained by highthroughput sequencing. A total of 4 986 sequences,which contained microsatellites and could be designed with primers were obtained. Twenty seven (11.30%) out of 239 microsatellite loci were proved to be polymorphic in 12 S. chuatsi populations. The number of alleles per locus ranged from 2 to 8 (5.63±1.84),the effective number of alleles per locus ranged from 1.86 to 6.80 (3.99±1.56),the observed heterozygosity per locus ranged from 0.21 to 0.78 (0.49±0.16),the expected heterozygosity per locus ranged from 0.46 to 0.85 (0.71±0.12),the polymorphic information content per locus ranged from 0.37 to 0.84 (0.66±0.15),and the gene flow per locus ranged from 0.11 to 2.14 (0.67±0.45). The number of alleles and the effective number of alleles per locus in the Jiayu population were the highest among 12 populations. The observed heterozygosity and the expected heterozygosity per locus in the Wuhan population were the highest. The Nei’s genetic distance ranged from 0.103 0 to 1.511 7,the Nei’s genetic identity ranged from 0.220 5 to 0.902 2. The maximum Nei’s genetic distance (1.511 7) and the minimum Nei’s genetic identity (0.220 5) were between the Fuyuan population and Shunde population. The minimum Nei’s genetic distance (0.103 0) and the maximum Nei’s genetic identity (0.902 2) were between the Fuyuan population and Shunde population. UPGMA cluster analysis showed that one branch of the Pearl River system and the other branch of the Yangtze River system gathered firstly and then converged with the branch of the Heilongjiang River. The genetic differentiation coefficient FST among the 12 groups was 0.041 80.611 0,and the genetic differentiation among the groups reached the significant level (P＜0.05).The minimum FST (0.041 8) was between the Liangzihu population and Wuhan population,and the maximum FST (0.611 0) between the Shunde population and Fuyuan population. AMOVA analysis showed that the genetic variation among populations accounted for 33.14%,and the genetic variation within populations was 66.86%. Structure analysis showed that 12 populations were divided into 5 subpopulations. This study could provide useful basic data for protection of germplasm resources and variety breeding in S. chuatsi.