Chlamydiosis, caused by Chlamydia pecorum, is regarded as an important threat to koala populations. Across the koala's geographical range, disease severity associated with C. pecorum infection varies, with pathogen diversity and strain pathogenicity being likely important factors. To examine C. pecorum diversity on a sub-population level a Multi-Locus Sequence Typing (MLST) scheme, containing the housekeeping genes; gatA, oppA_3, hflX, gidA, enoA, hemN and fumC, was used to type strains from two sub-populations of koalas from the Liverpool Plains, NSW, Australia, with different disease expressions. Typing of samples from 2015 to 2017, revealed a significant association between sequence type ST 69 and clinical disease and a significant difference in sequence type frequencies between sub-populations. Sequence type ST 69 has previously been identified in both subclinical and clinically diseased koalas indicating that these markers alone are not illustrative of pathogenicity. However, recent emergence of this sequence type in a naïve population may explain the differing disease expressions. Sequence types ST 73 and ST 69 have been described in koalas across a broad geographic range, indicating multiple introduction events and/or a limited veracity of the MLST loci to explore fine scale epidemiological investigations, particularly those examining the interface between pathogenic strain and disease outcome.