Abstract: A library rich in CA/GT microsatellites was constructed from the Paralichthys olivaceus genome by combining biotin capture method and radioactive labeling hybridization. Five hundred and twenty six positive clones were obtained through twice screens. Sequencing confirmed 133 microsatellite loci (number of repeats≥5) in 119 positive clones. Of these microsatellites, two (1.5%) had compound repeat motifs, 63 (47.37%) had perfect motifs and 68 (51.13%) had imperfect motifs. Primer pairs were designed in the flanking regions of 22 microsatelites and subjected to PCR amplification. In 8 artificial gynogenesis families, four pairs failed to amplification, one pair was monomorphic, and the rest were polymorphic with an average of 5.2 alleles per locus. Heterozygosities ranged between 0.375 and 0.846, PIC ranged between 0.305 and 0.823. The results suggested that most of the microsatellites we isolated were qualified to be applied to the population genetic studies of P. olivaceus.