We report four highly informative multiplex PCRs developed from twelve previously described ESTSSRs in Crassostrea gigas. We evaluated and validated these multiplex PCRs in twelve full-sib families. The average allelic richness and the polymorphism information content (PIC) were 11.1 and 0.811 respectively. The combined power of exclusion was greater than 99.99% using all four multiplex assays. A hundred and forty three tests of segregation ratios revealed 11 significant departures from expected Mendelian ratios. The frequency of null alleles was estimated as 4.9% of all the alleles segregating based on a within-family analysis of Mendelian segregation patterns. Parentage analysis of real offspring demonstrated that 97% of all offspring were unambiguously allocated to a pair of parents based on two multiplex PCRs with only a 4% error rate, and 100% of the offspring were correctly allocated to their parents when three multiplex PCRs were used.