A set of 72 microsatellite markers distributed evenly among 24 linkage groups were selected from the published genetic linkage maps of Japanese flounder Paralichthys olivaceus. In two normal diploid full-sib families, the test for Mendelian inheritance showed that genotypic segregation deviations were not significant at all analysed loci. To estimate microsatellite-centromere map distances, four meiotic gynogenetic diploid lines were produced by the activation of eggs using UV irradiated sperm of red seabream Pagrus major and cold-shock treatment to block the extrusion of the second polar body. Under the assumption of complete interference, 21 markers were located in the centromeric region, 39 in the telomeric region and the rest in the intermediate region of linkage groups. A total of 192 mitotic gynogenetic diploids from one spawn were identified by these markers. Genotype analysis showed that the number of homozygous individuals decreased as microsatellite-centromere map distance increased on each linkage group.