The objectives of this study were to characterize polymorphisms within the coding region of estrogen receptor beta (ERbeta) gene in a population of 57 female Japanese flounder (Paralichthys olivaceus) and to analyze the association of ERbeta polymorphisms with reproductive indices by polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP). Two single nucleotide polymorphisms (SNPs), SNP1 (c.577delC) and SNP2 [c.A891T (p.Gln114Leu)], were identified in the ERbeta gene. A one-way ANOVA revealed that SNP1 was significantly associated with the gonadosomatic index (GSI) in female Japanese flounder (P < 0.05). And SNP2 was significantly associated with the serum 17beta-estradiol (E2) level and GSI (P < 0.05). Individuals with genotype AB of SNP2 had significantly higher serum E2 level and GSI than those of genotype AA (P < 0.05). Moreover, the hepatosomatic index (HSI), a marker for genetic effects, was significantly higher for diplotype D2 compared with the other three diplotypes (P < 0.05). These results obtained in this study suggested that SNP2 could influence reproductive endocrinology of female Japanese flounder and be useful as a potential candidate genetic marker for the selection of reproductive indices in female Japanese flounder.