The DMRT family of transcription factors play a pivotal role in sex determination. In order to investigate the function of Dmrt3, a member of the DMRT family, in the embryogenesis and sex differentiation, as well as its potential implication in breeding of goldfish, we cloned the full length cDNA of Dmrt3 and analyzed its expression during the early embryogenesis and in various adult tissues in goldfish, Carassius auratus. The entire goldfish Dmrt3 cDNA is 2182 bp long, including a 408 bp 5￠-UTR, a 427 bp 3'-UTR and a 1347 bp open reading frame (ORF); the latter encoded a protein with 448 amino acids. Protein structural prediction based on the putative amino acid sequence of DMRT3 revealed that goldfish DMRT3 contained a common DM domain and a DMA domain, suggesting that it was more phylogenetically related to DMRT4 and DMRT5. Nested PCR examination indicated that the expression of Dmrt3 was undetectable until bud stage, and its expression obviously increased but still at a low level at 15-somite stage. In adult tissues, DMRT3 was expressed exclusively in the testes. This expression pattern suggested that Dmrt3 might regulate organogenesis and gonad development of male. No methylation of the Dmrt3 promoter CpG-island in gametes and in various tissues was observed using bisulfite sequencing analysis, suggesting that DNA methylation did not regulate the sex-specific and tissue-specific expression of Dmrt3. Moreover, we identified a pseudogene, named pDmrt3, from the reverse transcript of Dmrt3. These results provide essential materials for studying the function of Dmrt3 in the sex differentiation and potential application in breeding of goldfish, as well as the evolutionary relationship of Dmrt genes.