The Pallas squirrel, Callosciurus erythraeus, is an introduced species to Japan where a male-biased sex ratio has been reported at the weaning stage in the field. For further examination of their earlier (fetal) sex ratio, a new PCR primer set was designed to amplify a portion of the Sry (sex-determining region Y) gene. This successfully produced sex-specific PCR profiles for known-sex adult samples. Using this technique we could determine the sex of fetal samples dissected out of mothers shot as pests on Izu-Oshima Island, Tokyo, Japan. The fetal sex ratio was significantly male-biased in 2000, but not in other years. The sex ratio tending towards male-bias in this squirrel is discussed from a local resource competition theory.