In this study, we examined whether a homolog of the master sex-determining gene amhy of Odontesthes hatcheri is present and plays any role in testis determination of pejerrey O. bonariensis, a species otherwise known for its strong temperature-dependent sex determination (TSD). Screening of wild and laboratory-reared pejerrey for amhy revealed a high, although not complete linkage with phenotypic sex. The sex ratio in an amhy(+/-)/amhy(-/-) full sibling progeny reared during the thermolabile period of sex determination at an intermediate temperature of 25 degrees C was 68.7% male: 31.3% female; all amhy(+/-) fish developed as males whereas about 2/3 and 1/3 of the amhy(-/-) were female and male, respectively. Expression analyses revealed that amhy transcription began during embryo stage and decreased by the end of sex determination period. The autosomal amha was present in all individuals regardless of amhy genotype; its expression increased significantly from the end of the same period in the gonads of all amhy(+/-) but only in part of the amhy(-/-) animals. After histological gonadal differentiation, all gonads of amhy(-/-) animals with amha ISH signals were testes and those without it were ovaries. These results suggest that amhy is important for testicular differentiation in pejerrey, at least at intermediate temperatures. Thus, we hypothesize that amhy(+/-) animals differentiate as males by expression of either amhy alone or amhy and amha together whereas the amhy(-/-) probably rely solely on amha expression. These findings represent the first clear genomic evidence that genotypic and environmental sex determinants can coexist in species with marked TSD such as the pejerrey. The finding of amhy will make possible to monitor wild pejerrey populations for mismatches between genotypic and phenotypic sex and may prove instrumental for field studies addressing the effects of endocrine disruptors or abnormal temperatures on reproduction and the ecological relevance of TSD for this species.