The Chlamydiales are a unique order of intracellular bacterial pathogens that cause significant disease of birds and animals, including humans. The recent development of a Chlamydiales-specific 16S rDNA polymerase chain reaction (PCR) assay has enabled the identification of Chlamydiales DNA from an increasing range of hosts and environmental sources. Whereas the Australian marsupial, the koala, has previously been shown to harbour several Chlamydiales types, no other Australian marsupials have been analysed. We therefore used a 16S rDNA PCR assay combined with direct sequencing to determine the presence and genotype of Chlamydiales in five wild Australian mammals (gliders, possums, bilbies, bandicoots, potoroos). We detected eight previously observed Chlamydiales genotypes as well as 10 new Chlamydiales sequences from these five Australian mammals. In addition to PCR analysis we used antigen specific staining and in vitro culture in HEp-2 cell monolayers to confirm some of the identifications. A strong association between ocular PCR positivity and the presence of clinical disease (conjunctivitis, proliferation of the eyelid) was observed in two of the species studied, gliders and bandicoots, whereas little clinical disease was observed in the other animals studied. These findings provide further evidence that novel Chlamydiales infections occur in a wide range of hosts and that, in some of these, the chlamydial infections may contribute to clinical disease.