AbstractThe order Chlamydiales are biphasic intracellular bacterial pathogens infecting humans and domesticated animals. Wildlife infections have also been reported, with the most studied example being Chlamydia pecorum infections in the koala, an iconic Australian marsupial. In koalas, molecular evidence suggests that spill-over from C. pecorum infected livestock imported into Australia may have had a historical or contemporary role. Despite preliminary evidence that other native Australian marsupials also carry C. pecorum, their potential as reservoirs of this pathogen and other Chlamydia-related bacteria (CRBs) has been understudied. Mucosal epithelial samples collected from over 200 native Australian marsupials of different species and geographic regions across Australia were PCR screened for Chlamydiales. Previously described and genetically distinct C. pecorum genotypes and a range of 16S rRNA genotypes sharing similarity to different CRBs in the broader Chlamydiales order were present. One 16S rRNA Chlamydiales genotype recently described in Australian ticks that parasitise native Australian marsupials was also identified. This study provides further evidence that chlamydial infections are widespread in native fauna and that detailed investigations are required to understand the influence these infections have on host species conservation, but also whether infection spill-over plays a role in their epidemiology.