Some biomedical research procedures using laboratory mice, such as the preparation of sex-specific fetal cell cultures, require the sex of fetuses and newborn pups to be determined. Although neonate mice can be sexed anatomically on the basis of the anogenital distance (AGD), 48% of newborn pups are reported to be unclassifiable using this method (1). The existing molecular methods for sexing are PCR-based assays that combine two pairs of primers together in a multiplex reaction to amplify the Y-chromosomespecific gene Sry and an autosomal gene—either Il3 (chromosome 11) or Tshb (chromosome 3)—that serves as an internal control of PCR amplification (2, 3). Here we present a novel PCR assay that uses only one pair of primers in a single reaction tube to simultaneously amplify DNA fragments from both the X-and Y-chromosomes. Using the Sequencher™ 4.1 software (Gene Codes Corporation, Ann Arbor, MI, USA), we aligned DNA sequences of the X-chromosome-specific gene