Vertebrates exhibit a surprising array of sex-determining mechanisms, including X- and Y-chromosome heterogametes in male mammals, Z- and W-chromosome hetero-gametes in female birds, and a temperature-dependent mechanism in many reptiles1. The Y-chromosome-linked SRY gene initiates male development in mammals2,3, but other vertebrates lack SRY and the genes controlling sex determination are largely unknown. Here we show that a gene implicated in human testis differentiation, DMRT1, has a gonad-specific and sexually dimorphic expression profile during embryogenesis in mammals, birds and a reptile (Alligator mississippiensis). Given the different sex-determining switches in these three groups, this gene must represent an ancient, conserved component of the ver-tebrate sex-determining pathway.