FOXL2 which is a putative winged helix/forkhead transcription factor gene and a sexually dimorphic marker of ovarian differentiation plays an important role in ovarian development, granulosa cell differentiation, and thus the proper maintenance of ovarian function. The aims of this study were to characterize polymorphisms within the FOXL2 gene in a population of 52 female Japanese flounder and analyze the association of FOXL2 polymorphisms with reproductive performance by polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP). Results indicated that five single nucleotide polymorphisms (SNPs), which were SNP1 [c.540A>C (p.Asn102His) and c.591A>G (p.Asn119Asp)], SNP2 [c.864G>A (p.Lys210Glu)and c.875G>A] and SNP3 (c.1169C>A), were identified in the FOXL2 gene. General Linear Model (GLM) analysis showed that SNP1 in the forkhead domain was significantly associated with gonadosomatic index (GSI) (P<0.05). SNP2 in the downstream of forkhead domain was significantly associated with serum 17beta-estradiol (E(2)) level (P<0.05). And SNP3 in the 3'-UTR was significantly associated with hepatosomatic index (HSI) (P<0.05). Moreover, the evaluation of the genetic effects for both Testosterone (T) level of diplotype D3 and GSI of diplotype D5 suggested they were significantly higher than those of other four diplotypes (P<0.05), respectively. These results implied that these SNPs could influence reproductive endocrinology of female Japanese flounder and be also used in marker-assisted selection (MAS) program to reproductive performance in female Japanese flounder in the future.