Olive flounder(Paralichthys olivaceus)is one of economically important fish in China.It is distributed widely over the Bohai Sea,the Yellow Sea and the South Sea in China,North Korea,Japan and along the east coastline of Russia.As its natural resource is exhausting gradually,olive flounder has become an important cultured fish in China.Polymorphic available DNA markers are needed for its genetic research.Microsatellites recently have become an extremely popular marker in a wide variety of genetic investigations.In this research,genomic DNA was extracted from fin of olive flounder collected from one locality along the Chinese coast.The genomic DNA was digested by using both RsaⅠ and Bst UI polymerase.And then a microsatellite-enriched genomic library was constructed.By using(GA)13 as probes,177 positive clones were identified from 269 clones,and 191 microsatellites were found.Among the 191 microsatellites,74.35% were perfect,14.66% were imperfect and 10.99% were compound type.50 markers were assessed using 32 individuals.Of the 50 loci 31 were found to be polymorphic.The result showed that the number of alleles ranged from 2 to 9,with an average of 3.939 4 alleles per locus.The number of effective alleles per locus is 3.052 2. The values of observed and expected heterozygosity per locus is 0.650 1 and 0.586 6, respectively.The values of Hardy-Weinberg equilibrium ranged from 0.012 587 to 0.984 917.These markers will be useful for population structure assessment,molecular marker-assisted selection and genetic linkage map construction for this species.