A novel method for sex identification, using a denaturing high-performance liquid chromatography (DHPLC) system, is described. Among many methods for identifying sex, the most popular and credible system has been the polymerase chain reaction (PCR) method, using nucleotide primer sets of the amelogenin gene, which is shared on both the X and Y chromosomes. With this conventional method, the judgment depends on detection of the size difference between the PCR products derived from the X and Y chromosomes. In this study, we adopted DHPLC to detect the difference by checking heteroduplex formation between the products, which enabled us to shorten the PCR products to 45bp and the separation time to within a period of 8min per sample. This new system may have wide applications in many different fields, such as forensic medicine, prenatal diagnosis, inbreeding of animals, and anthropology.