Iwagaki oyster, Crassostrea nippona, widely distributes along the seashore of Eastern Asia, and has been considered to be a potential breeding species due to its delicious taste and high commercial value. In order to study its genetic background and population structure, we developed 46 novel polymorphic microsatellite markers using next-generation sequencing technique and characterized them in 30 individuals. The number of alleles ranged from 3 to 22, while the observed and expected heterozygosities varied from 0.133 to 1.000 and 0.455 to 0.949, respectively. Fifteen microsatellite markers were selected and grouped into five highly informative multiplex PCRs for C. nippona. We evaluated and validated these multiplex PCRs in a cultured population including 173 candidate parents and 486 offspring. In actual parentage analysis, 80% of the offspring were correctly assigned to their parental pairs using three multiplex PCRs. Furthermore, the success rate of parentage assignment reached 96% when the other two multiplex PCRs were added. These 46 microsatellite loci with high variability and the five multiplex PCRs described here provide a powerful tool for pedigree reconstruction, resource conservation and selective breeding program of C. nippona.