Twenty-eight polymorphic microsatellite markers for Japanese sea urchin (Strongylocentrotus intermedius) were developed from the EST database of a related species purple sea urchin (S. purpuratus). The characterization results showed the moderate polymorphisms at these loci. The number of alleles ranged from 2 to 5, and the values of H_o and H_c varied from 0.0667 to 0.9655 and 0.0655 to 0.7350, respectively. No significant linkage disequilibrium (LD) between pairs of loci was found and 21 loci conformed to the Hardy-Weinberg equilibrium (HWE). The results in this study well demonstrated the feasibility of microsatellite marker development via mining EST database of a genetically related species.