Abstract: In the present study, microsatellites DNA was isolated and enriched from genome of sea cucumber (Apostichopusjaponicus) by magnetic beads 123 positive clones and 106 microsatellite sequence were obtained. The microsatellite sequences were structurally categorized into follows: perfect 48 (45.28%), imperfect 39 (36. 79% ) ,and compound 19 ( 17.92% ). Besides the CA/GT contained in the oligoprobe, CATA, CT,CCA,GA, CACT, CAA, and CACAGA repeats were found. Among the microsatellite repeats, relatively short arrays were abundant, the repeating frequence of 3 - 10, 11 - 18 and ≥35 accounted for 39.62%, 26. 42% and 16.98%, respectively. The largest array contained 148 repeats. This study will enrich microsatellite markers of sea cucumber. Base on the flanking sequence of the microsatellite loci, 22 pairs of microsatellite primer were designed and applied to investigate the polymorphism in populations of Chinese and Russian sea cucumber populations. 17 pairs of primer generated PCR amplification products, and 7 of them yield polymorphic products that can be used in the study of population genetics and genetic mapping.