The barfin flounder has been recently declared to be rare, as the number of wild individuals in Japan has greatly reduced since 1975. The Japanese National Center for Stock Enhancement started the stocking program for this species on 1987. In the present study, microsatellite DNA loci were applied in order to determine the pedigree of the hatchery-produced juveniles for stock enhancement. Additionally, using six polymorphic microsatellite markers, the accuracy to assign dyads to categories of kinship was estimated using the relatedness estimator (RXY), similarity indexes (individual genetic identity [GI] and proportion of shared alleles [PS]), identification of kin groups using unweighted pair group method with arithmetic mean (UPGMA) clustering based on interindividual genetic distances (1-RXY, 1-PS, and 1-GI), a likelihood ratio approach, and partition methods to separate individuals into cohorts. The results were compared with the pedigree relationship previously obtained from parentage analysis. Estimation of the kinship relationship between individuals could be better inferred by means of UPGMA dendrogram clustering based on 1-GI or 1-PS as interindividual genetic distances, or using computer software to perform partitioning of set of individuals.