We have developed a PCR-based protocol to determine the gender of tissue samples originating from elk (Cervus elephus canadensis), moose (Alces alces) and white-tailed deer (Odocoileus virginianus). The technique simultaneously amplifies a conserved region of the sex-determining gene on the Y-chromosome (Sry) and a region of the Fragile X mental retardation gene (Fmr-1). The multiplex nature of this protocol allows the determination of gender using the Sry marker with the Fmr-1 marker providing an internal control. This technique is applicable to the enforcement of the validation tag system for game species. Data are provided from a wildlife investigation in Ontario.