Abstract: Eighteen microsatellite markers were analysed with a paternity testing application using 26 mitogynogenetic Japanese flounder (Paralichthys olivaceus) progeny and their six assumed female-parents. The amplified products of PCR were analyzed by 8% polyacrylamide gel electrophoresis. Electrophoresis patterns showed that only one pair of primers had a monomorphic pattern, and the others were polymorphic. Cumulate exclusion probability of polymorphic loci was 0.9985 and cumulate power of discrimination was 0.9999. An effective method was applied to track the gynogenetic maternal parents of 26 progeny. The genotypes of six progeny were not completely identical to their mothers on some loci. The construction of pedigree was perfected by looking at microsatellite loci from paternity tests which are essential to the deeper analysis of gynogenetic Japanese flounder.