To carry out accurately the parentage determination on different families in Japanese flounder and select the microsatellite DNA markers with higher parental probabilities of exclusion,in this study,10 mature females and 10 mature males chosen from the selection and breeding founder were used to produce 10 full-sib families of Japanese flounder.30 individuals from each family separately raised were random selected to construct the family population whose pedigree structure was known.200 individuals from the families raised together were random selected to construct the mixed population whose pedigree structure was unknown.A set of 48 microsatellite DNA markers distributed evenly on 24 linkage groups were selected from the second genetic linkage maps of Japanese flounder,furthermore,2 markers were located on each linkage group.The results showed that 10 microsatellite DNA markers took on the higher parental probabilities of exclusion and richer genetic polymorphism based on the genetic analysis of family population,and their range for Excl 1 and Excl 2 was from 0.655 to 0.719 and from 0.792 to 0.837,respectively.The combined probabilities of exclusion gradually increased as the number of markers identified increased.It reached 100% when 8 microsatellite DNA markers were jointly used.The results of parentage determination of mixed population by using these markers demonstrated that 13 females and males contributed to the reproduction in total.To some extent,there were differences in the number of offspring from the combination of different parents.Moreover,significant differences did not exist in each genetic statistical data between the parental and progeny populations.The research demonstrated that these microsatellite DNA markers selected with higher parental probabilities of exclusion can effectively perform the parentage determination on family in Japanese flounder,therefore,they are designated as candidate markers to develop joint breeding by combining the family selection with DNA molecular marker in future.