Cytochrome P450 aromatase, which is encoded by the CYP19a gene, converts androgens to estradiol. Considerable evidence suggests that estrogens play an important role in fish reproductive process. Therefore CYP19a is an excellent candidate gene for reproductive traits. Variants in the promoter of the CYP19a gene might also be involved in the control of aromatase expression and affect regulatory mechanism linking cholesterol metabolism to the synthesis of sex steroids. In this study, nine single-nucleotide polymorphisms (SNPs) were detected with polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP), namely A-680G, G-672A, AGTAGT-649 inserting or deleting, T-623C, C-410A, T7-454A, T-402C, TTTCCAGACTGA-345 inserting or deleting, and G-297C. Nine SNPs within the promoter of the CYP19a gene were tested for association with four reproductive traits [serum testosterone (T), serum 17beta-estradiol (E(2)), hepatosomatic index (HSI), and gonadosomatic index (GSI)] in a population of 50 female Japanese flounder individuals. A locus, P3 (TTTCCAGACTGA-345 inserting or deleting, G-297C), was significantly associated with 17beta-estradiol (E(2)) level (P < 0.05) in female Japanese flounder. In addition, there was significant association between one diplotype based on nine SNPs and reproductive trait. The genetic effect for E(2) level of diplotype D3 was significantly higher than those of other diplotypes (P < 0.05). Results indicate that these genetic effects of those variants on E(2) level may help to explain CYP19a gene status in the reproductive endocrinology of Japanese flounder.