Road-killed animals are a readily available but often overlooked source of specimens and data. Determining the sex of road-killed possums is often difficult due to the severe damage carcasses sustain on impact with vehicles. Consequently, valuable information is lost. To assess the potential application of genetic techniques to address this problem, tissue biopsies (n=47) taken from road-killed possums were sexed via PCR, through the simultaneous use of W- (G6PD) and Y-linked (SRY) primers. Comparisons with animals of known sex were used to validate the accuracy of this method, with 100% (n=19) of samples correctly identified in a blind test. This methodology can provide researchers with valuable, additional data for road-kill studies where assigning sex in the field is not possible.