In this research,we have analyzed the genetic diversity of wild and cultured populations of 28 polymorphic microsatellite markers for Japanese sea urchin (Strongylocentrotus intermedius) via mining EST database of relative species (S. purpuratus). S.intermedius belongs to Echinodermata, Eleutheroze and Echinodea, which is distributed in the northeastern Japan, the Korean Peninsula and the Far East coast of Russia. It was introduced into China by Dalian Fisheries University in 1989 and has been breeding for more than 6 generations. ESTSSRs recently have been commomly used in genetic investigations. The flanking sequence of ESTSSRs is so highly conserved that the primers can be tansferred between similar species. Therefore, in this experiment, the primers were designed with the EST sequence of S. purpuratus from the database in GenBank (http://www.ncbi.nlm.nih.gov/dbEST/index.html), then polymerase chain reaction (PCR) was used to identify the effective primers that matched with the S.intermedius which was related to S. purpuratus. Results showed: A total of 91 different alleles were found and the number of alleles in each locus ranged from 2 to 6．In the 2 populations，the average number of alleles was both 3.071 4; the number of mean valid alleles was 2.231 9 and 2.227 1 respectively; the value of average observed and expected heterozygosity were 0.523 4，0.536 5 and 0.486 8，0.499 3 respectively; the mean PIC was 0.447 7 and 0.439 6. The difference in genetic diversity among the 2 populations was not significant. The genetic simi1arity coefficient of the 2 populations was 0.758 7，and the genetic distance of the populations was 0.276 2. Chisquare test was used to analyze the genotypes based on HardyWeinberg equilibrium，the P value showing that 50% loci departed from HardyWeinberg equilibrium. The results of the Ftest suggested that both the two populations have surplus heterozygous, and there is no obvious inbreeding phenomenan in the two populations. The results also displayed low genetic differentiations between the 2 populations，and the variance was mainly due to individual difference among the populations. Although the cultured population has been breeding for several generations, there is no significant difference on genetic polymorphism with the wild population.