Ross River virus (RRV) is a mosquito-transmitted Alphavirus emerging in urban centres throughout Australia. The Common brushtail possum (Trichosurus vulpecula), a native marsupial that has successfully adapted to human settlement, has been implicated as a maintenance reservoir for RRV. In the present study, RRV exposure was assessed amongst 72 urban-adapted possums from Northern Sydney and ten possums from a woodland area, remote from urbanisation. Serological screening was performed using an enzyme-linked immunosorbent assay to detect RRV antibodies in possum sera. Findings indicated that both possum populations from urban and woodland habitats were negative for the presence of RRV antibodies. Lack of exposure to RRV highlights that the host status of possums is contingent upon factors other than their abundance and proximity to human settlement. In view of the potential for climate change to favour transmission of mosquito-borne disease in Australia, identification of wildlife populations entirely absent of RRV may prove useful for monitoring the predicted spread of the virus.