The mature spawners selected from Japanese flounder selection and breeding founder were used to produce the normal diploids (ND), meiotic gynogenetic diploids (MGD-1), successive two generation meiotic gynogenetic diploids (MGD-2) and inbred diploid (MGD1H) by crossing mature female from MGD-1 with induced sex reversed male. A set of 72 microsatellite markers distributed evenly on 24 linkage groups were selected from the published genetic linkage maps of Japanese flounder. To estimate microsatellite-centromere map distances, four MGD-1 lines were produced. Under the assumption of complete interference, 17 markers were located in the centromeric region, 36 in the distant region from the centromere and 19 in the intermediate region of linkage groups. The genetic characteristics of four types of diploid in Japanese flounder were identified by these markers. Analysis showed that the number of allele (A) and polymorphism information content (PIC) have limited variation range, in which, the A and PIC of ND were the highest while those of MGD1H were the lowest. In four types of diploid, the observed heterozygosity decreased and homozygosity increased as microsatellite-centromere map distance increased. The highest and lowest percentages of homozygous individuals were found in centromeric region from 8.8% to 29.1% and in telomeric region from 2.4% to 23.2%, respectively. In which, the variation range of MGD-1and MGD-2 were significant higher than that of the rest diploids. Thus, choice of microsatellite markers located on different positions in linkage group have greatly impact on identifying genetic characteristic of different diploids in Japanese flounder.