Abstract Hendra virus (HeV) continues to cause fatal infection in horses and threaten infection in close-contact humans in eastern Australia. Species of Pteropus bats (flying-foxes) are the natural reservoir of the virus. We caught and sampled flying-foxes from a multispecies roost in southeast Queensland, Australia on eight occasions between June 2013 and June 2014. The effects of sample date, species, sex, age class, body condition score (BCS), pregnancy and lactation on HeV antibody prevalence, log-transformed median fluorescent intensity (lnMFI) values and HeV RNA status were assessed using unbalanced generalised linear models. A total of 1968 flying-foxes were sampled, comprising 1012 Pteropus alecto, 742 P. poliocephalus and 214 P. scapulatus. Sample date, species and age class were each statistically associated with HeV RNA status, antibody status and lnMFI values; BCS was statistically associated with HeV RNA status and antibody status. The findings support immunologically naïve sub-adult P. alecto playing an important role in maintaining HeV infection at a population level. The biological significance of the association between BCS and HeV RNA status, and BCS and HeV antibody status, is less clear and warrants further investigation. Contrary to previous studies, we found no direct association between HeV infection and pregnancy or lactation. The findings in P. poliocephalus suggest that HeV exposure in this species may not result in systemic infection and virus excretion, or alternatively, may reflect assay cross-reactivity with another (unidentified) henipavirus.