A new set of 34 microsatellite markers was isolated and characterized in pearl oyster Pincada martensi from a CA16-enriched genomic library. Of 60 microsatellite markers screened in a wild population which contained 30 individuals, 34 markers occurred unambiguous bands, including seven monomorphic loci, 5 primer pairs (HHM844, HHM852, HHM888, HHM891, HHM899) were duplicated. These polymorphic markers showed high genetic variability represented mainly by the number of alleles, which ranged from 2 to 6, the expect heterozygosity which ranged from 0.1653 to 0.7622, and the polymorphic information content which ranged from 0.1516 to 0.7279. These markers could increase the available molecular resources that can be used to analyze genetic diversity and structure of pearl oyster.