The analysis of parentage using microsatellite markers is of increasing importance, notably in aquaculture genetic research where communal rearing of mixed families can be used to reduce unwanted environmental variance. We present here an optimization of parental genotype assessment for larvae or adults of the Pacific oyster, Crassostrea gigas, using a multiplex system of three microsatellite loci. In conjunction with a simple DNA extraction protocol, this method enables high throughput analyses of parentage in C. gigas. Using this method, we successfully determined the parentage of 93% (1224 out of 1318) of the progeny in a factorial cross between 3 females and 10 males. The inability to genotype the remaining 7% was due to DNA degradation of larvae rather than assignment uncertainty.