It is important to consider the difference in contribution to reproduction between parents when assessing the effectiveness of enhancement programs. We used 18 microsatellite markers to estimate the of thirteen parents (five ♀, eight ♂), based on a sample of one hundred and eighty eight offspring of Japanese flounder ().When using 4 microsatellite markers, the PE was0.999 9 and accuracy was 92.02%. Using 6 microsatellite markers, the PE was 0.999 999 and accuracy was 96.81%. When using 8 microsatellite markers, the two values were 0.999 999 999 and 97.87%; using 10 microsatellite markers, the accuracy was 99.47%, and between 12–18 microsatellite markers, the accuracy was 100%. Our data suggest that the accuracy increased with an increase in the number of microsatellite markers. These SSR markers can be used for parentage determination and evaluating the effectiveness of enhancement releases. The results of PE testing suggest that paternity test accuracy of 100% can be achieved. Although all thirteen parents contributed to reproduction, there was considerable variation in contribution among individuals. The highest and the lowest parental contributions were 47.34% and 0.53%, respectively. The reasons driving these differences require further research.