The DM domain gene family has at least eight members with conserved DNA-binding DM domain, which encodes putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of Caenorhabditis elegans. Although some of the DM genes are involved in sexual development, the function of most of these genes remains unclear. There is also few structural and functional analysis concerning DM domain genes of the model fish, zebrafish. We report here molecular cloning, sequence, and expression of zebrafish Dmrt5, which consists of two exons, and encodes a 440-amino acid protein with conserved DMA and DMB domains in addition to DM domain. Phylogenetic analysis shows that zebrafish Dmrt5 fits within the Dmrt5 clade of fish and mammals. Zebrafish Dmrt5 was expressed in early gastrula period, subsequently increased to a high level in late stage of gastrula period (bud stage) and lower until the hatch period. In situ hybridization analysis showed its expression in developing central nervous system of embryos, especially in mid-brain and mid-hind brain boundary. In adult, its expression was restricted in brain and developing germ cells, especially in spermatogonia, spermatocytes, spermatids, and sperm cells, and in developing oocytes, including early perinucleolus stage oocyte, late yolk vesicle stage oocyte, and oil drop stage oocyte. These data suggest that zebrafish Dmrt5 have potentially important roles in gonadal development and may have contributed to the functional endocrine axis.