Oliver flounder, Paralichthys olivaceus, is not only a model animal for fish metamorphosis, but also an important cultured species in China. In this study, 42 unique expressed series tags (ESTs) containing 46 microsatellites were found from the cDNA library of premetamorphosing flounder. Twentyone microsatellite loci were tested in 30 individuals in the breeding population of P.olivaceus and 20 among them had polymorphism. The number of detected alleles varied from two to six, while the number of observed heterozygosity varied from 0.06 to 1.00, and the expected heterozygosity ranged from 0.059 7 to 0.825 2. The average of polymorphism information content (PIC) is 0.4575. The results indicate that these microsatellite loci will be useful for linkage map construction and markerassistedselection program of P.olivaceus. Three microsatellite loci with trinucleotide repeats buried in the coding region of genes were found to have polymorphism, which might be related to the gene function.