Genomic DNA was extracted from a male Japanese medaka (Oryzias latipes) and a DNA fragment of 1 908 bp in length was cloned using specific primers by polymerase chain reaction (PCR) amplification. DMY- gene was then identified after sequencing, which consisted of three exons and two introns and encoded a peptide of 150 amino acids. In our study, an accurate and effective method was established for sex identification (gene type) of any development stage of medaka, which is amplifying household βactin gene to testify the DNA template and PCR amplification, and then amplifying specific DMY-gene to distinguish the gender of medaka. The method has been successfully applied to sex identification in randomly selected mature medaka and new born fish eggs. 