Many animal species, including macropods, have the potential to act as atypical reservoirs of the causative agent of Q fever, Coxiella burnetii. The objective of this study was to determine the seroprevalence of C. burnetii in various macropod species in Australia. Competitive and indirect ELISAs were developed for the testing of macropod sera for antibodies to phase II and I C. burnetii antigens separately. A total of 500 macropod serum samples from selected species sampled in eastern and western coastal states of Australia were screened for the presence of anti-C. burnetii antibodies. An overall seroprevalence of 20.8% (95% CI 20.8–20.9%) was observed with 30.4% (30.2–30.9%) in northern Queensland, 13.0% (12.9–13.1%) in southern Queensland, 7.1% (7.1–8.0%) in western Queensland and 22.8% (22.7–22.9%) in south-western Western Australia. These data indicated that macropods represented a potential reservoir for zoonotic transmission of C. burnetii to domestic animals and the human population.