Japanese scallop is one of the most important cultured seashells in the world. Following about 20 years’ artificial culture, we need DNA markers for genetic research. Microsatellites recently have become popular markers in a variety of genetic investigations. In this research, we isolated microsatellite DNAs from the Japanese scallop Patinopecten yessoensis genome with magnetic beads. As a result, 136 positive clones were identified from 192 clones,and 179 microsatellites were found. Among the 179 microsatellites, 50.8% were perfect, 43.0% were imperfect and the rest were compound type (6.1%). Finally, 85 microsatellite primers were designed by software Primer Premier 5.0, and 40 pairs were screened; 15 loci showed polymorphism. In these microsatellite loci, genetic diversity was analysed in 2 cultured populations. The average number of allele (A) ranged from 3.27 to 3.40; the average heterozygosity (H_o) in 2 populations was 0.447 to 0.540; average excepted heterozygosity (H_e) was 0.468 to 0.493; and average polymorphism information content (PIC) was 0.443 to 0.463. The genetic distance was 0.1155 and genetic identity was 0.8909. Thirteen microsatellite loci except HLJX-028 and HJLX-031 could be used for genetic