Three third-generation of successive meiogynogenetic families (Meio-G3-1, Meio-G3-2 and Meio-G3-3) in Japanese flounder (Paralichthys olivaceus) were induced by artificially meiogynogenesis, which the eggs were activated by UV-irradiated red sea bream (Pagrus major) sperm, and followed with the cold-shock treatment. We evaluated the genetic structure of these three families using 30 microsatellite markers, and the efficiency of successive meiogynogenesis for producing highly inbred line was quantified by calculating the homozygosity and genetic similarity. The numbers of alleles detected were 35, 38 and 37 in Meio-G3-1, Meio-G3-2 and Meio-G3-3, respectively. And for these three families studied, the average numbers of alleles (A) were 1.17, 1.27 and 1.23; the average observed heterozygosity (H_o) were 0.1467, 0.2556 and 0.2056; the average homozygoisty among the 30 analyzed loci were 0.8533, 0.7444, and 0.7944. For the genetic similarity, the average similarity index between offspring within family was 0.9913, 0.9918 and 0.9838; between dam and offspring was 0.996, 0.9968 and 0.9923; and between families was 0.9717 (Meio-G3-1 and Meio-G3-2), 0.9810 (Meio-G3-1 and Meio-G3-3) and 0.9714 (Meio-G3-2 and Meio-G3-3), respectively. Our results indicate that the artificially induced successive meiogynogenesis can increase the homozygosity of individual, and also the genetic similarity of offspring within family and between families. And therefore, the artificially induced successive meiogynogenesis is a good method to establish inbred lines in fish.