Four genes related to the process of sex differentiation were cloned and their expression analyzed during gonadal differentiation of Odontesthes hatcheri. SF1, P450aro and AMH presented one single copy whereas Dmrt1 presented three isoforms. Among the four genes, clear sexually dimorphic expression was observed for P450arom and AMH, with increased expression of these genes in females and males, respectively. Furthermore, such sex differences were observed before the onset of ovarian differentiation, suggesting an important role for these genes during gonadal differentiation in this species.