Gynogenesis was considered to be a useful method to generate clonal lines in fish. Mitogynogenesis has been successfully applied to several fishes. In this study, mitogynogenesis was carried out in , by fertilizing eggs with UV-irradiated heterologous sperms of red sea bream, One hundred and seventy three mitogynogenetic diploids belonging to different families have been mixedly raised for 24 months. Eleven microsatellite markers were used to examine homozygosity and genetic relationship of mitogynogenesis in Japanese flounder. The results showed that among the 173 mitogynogenetic diploids 111 individuals were completely homozygous at all 11 loci, andThe number of the alleles (A) ranged from to eight , and average number of allele was 5.033 6. Effective alleles () were from 2.042 1 to 5.126 8and average number of effective alleles was 3.281 5. The genetic distance of 111 homozygous individuals was calculated and the cluster analysis was carried out by NJ method. All individuals were pooled into four groups. The results revealed that residual heterozygosity still remained in mitotic gynogenetic diploids. Heterozygous fish was obtained through mitogynogenesis as a result of the presence of meiogynogens. Therefore, precise assessing the homozygosity of mitotic gynogenetic diploids is necessary for successful clonal lines.