A repeat-enriched genomic library was constructed from southern flounder Paralichthys lethostigma in order to evaluate the level of genetic diversity and population structure. In the first batch, 45 positive clones were successfully sequenced and 43 sequences were found to contain repeat motifs. According to Weber(1990) classification rules, the sequences were divided into three categories: 26 perfect repeat sequences without interruptions in the runs of CA or GA dinucleotides (60.5%), 14 imperfect repeat sequences with one or more interruptions in the run of repeats (32.6%), and 3 compound repeat sequences with adjacent tandem simple repeats of a different sequence (6.9%). Fourteen pairs of microsatellite primers were selected and used to analyze the polymorphism and genetic structure of the wild and cultured population of southern flounder from America. The results were shown as follows: two loci were monomorphic, and 12 loci were polymorphic, in which 7 loci showed high polymorphic levels judged by PIC value (PIC>0.5).The averages of observed and expected heterozygosities of the 12 polymorphic loci were 0.36 and 0.57, respectively. In addition, a total of 90 alleles (50 effective alleles) were detected at 12 loci in total samples. The Hardy-Weinberg equilibrium (HWE) analysis showed that 9 loci deviated from genetic equilibrium to different extent in the two populations and heterozygote deficiency occurred in total samples (Fis>0). The level of genetic diversity in the wild population was significantly higher than that of the cultured population, and the genetic distance between two populations was 0.111 5, which indicated that the genetic differentiation between two populations was quiet weak. Twelve polymorphic microsatellites were identified in this study, and these microsatellite loci would be usful to analyze genetic diversity, and protect germ plasm resource of southern flounder.