Sex determination consists of somatic and germ-line sex differentiation hierarchies whose interaction is poorly understood. A single gene known to control somatic sex determination, the DM-domain containing (Doublesex/Mab-3 DNA-binding motif) gene, is highly conserved across species. Vertebrate DIMRT1 (DM-related transcription factor 1) expression occurs predominantly in testis. We, however, isolated two distinct DM-domain cDNAs from tilapia testis and ovary, named tDMRT1, and tDMO (DM-domain gene in Ovary), respectively. Despite high homology in the DM-domain, there is little similarity outside the DM-domain. A male specific motif is absent in tDMO indicating a similarity with the female type of doublesex in Drosophila. In contrast to the alternatively spliced male and female types of doublesex, tDMRT1 and tDMO cDNAs are encoded by two different genes. The mutually exclusive nature of tDMRT1 and tDMO expression in the testis or ovary suggests that they both play important roles in gonadal development and/or function.