, a common freshwater fish in China, were selected to determine the effects of an aromatase inhibitor on sex differentiation and related gene expression. Two-day-old juvenile were fed different doses of the aromatase inhibitor, letrozole (50, 100, and 200 μg/g diets) for 30 days. The effects of letrozoleon sex ratio, gonad histology, and expression were examined by morphological observation, histology, and real-time fluorescent quantitative PCR during the period of sex differentiation (12−30 d after hatching). The results showed that letrozole doses of 100 μg/g and 200 μg/g produced more males (65.9% and 71.3%, respectively) than did a dose of 50 μg/g (64.7%) (<0.05). However, no significant difference in sex ratio was observed between the 50 μg/L 17α-MT treated group and the control group (51.1%). In addition, letrozole accelerated the occurrence of primary spermatocytes for 2 and 5 days, but deferred that of ovarian cavity and primary oocytes for 3 and 6 days, respectively. Both Cyp19a1 expression prior to and post sex differentiation (12−30 d after hatching). However, In addition, letrozole at dose of 200 μg/g inhibited the expression of both Cyp19a1. Our results suggest that letrozole doses above 100 μg/g could induce involved in the