Abstract: The molecular mechanism of temperature-dependent sex determination of Mauremys mutica remains unclear. To investigate the role of Dmrt1 in male sexual differentiation in Mauremys mutica, we cloned the full-length cDNA of Dmrt1 by RACE PCR and investigated its expression. The sequence of Dmrt1 was 1811 bp in length, containing a 572 bp 3′ untranslated region (UTR) and an 1110 bp open reading frame (ORF) encoding 370 amino acid residues. The maximum homology between the derived Dmrt1 of Mauris and Mauremys reevesi Dmrt1 was 97.57%, and the maximum homology to mouse Dmrt1 was 63.25%, and the maximum to Acipenser sinensis Dmrt1 was 21.62%. The reverse transcription quantitative real-time PCR (RT-qPCR) revealed that Dmrt1 was abundantly expressed in adult testis, low in adult ovary and hardly expressed in all other somatic tissues. Importantly, the expression levels of Dmrt1 at 16—25 phase of androgenetic temperature increased gradually and were significantly higher than those of gynogenetic temperature. Chemical in situ hybridization showed that Dmrt1 mainly expressed in the sertoli cells, and was detected modestly in spermatogonia, primary spermatocytes and secondary spermatocytes, but was hardly detected in sperm cells and somatic cells in testis. These findings indicated that Dmrt1 gene may be involved in sex determination and male gonadal differentiation in Mauremys mutica. This study provides a foundation for understanding the mechanism of sex determination in Mauremys mutica.