Chlamydia-related disease has a detrimental effect on Australia's free-range koala (Phascolarctos cinereus) populations. The chlamydial species responsible for ocular, urogenital and respiratory disease in the koala have previously been identified as Chlamydophila pecorum and Chlamydophila pneumoniae. Epizootiology studies have therefore used species specific PCR assays to detect chlamydial infections. In the current study, we used a broad range PCR amplification and cloning strategy to identify all strains of Chlamydiales in the koala. Sequencing of 16S rRNA gene PCR products, cloned from Chlamydiales--order positive swab samples identified nine novel koala Chlamydiales genotypes, including multiple novel chlamydial genotypes present in a single sample. The novel koala genotypes are clustered together with other Chlamydia-like bacteria within a second lineage separate from the known Chlamydiaceae species. Two new primer sets UKC-A and UKC-B were designed to detect five of the nine novel Chlamydiales and were applied to swab samples collected from two wild koala populations. Using these new UKC PCR assays, UKC-A type Chlamydiales sequences were more prevalent (72%; 18/25) compared to UKC-B (24%; 6/25). UKC sequences were most commonly found as dual infections with C. pecorum. This report provides the first description of additional members of the order Chlamydiales infecting the koala.