Chlamydia are considered to be the most important pathogen of koalas in which they cause ocular and urogenital infections. As recently as 1996 it was realised that koala chlamydial infections do not belong to the species Chlamydia psittaci but instead should be reassigned to the species C. pecorum and C. pneumoniae. We have used DNA sequence analysis of part of the chlamydial major outer membrane protein gene, ompA VD4, to compare 15 koala C. pecorum isolates. Unexpectedly, we found that the koala isolates did not cluster as a single branch in the C. pecorum tree, but instead were represented by five genetically very distinct genotypes. Two of the genotypes (which contained five koala isolates each) were koala-specific whereas one genotype contained a single koala isolate plus three sheep and two cattle isolates. For all five koala genotypes, their nearest relatives were not other koala genotypes, but sheep, cattle or pig isolates. It may be inferred from our data that C. pecorum strains infecting koalas do not form a monophyletic group with respect to other C. pecorum strains, and therefore the model which states that there was a single acquisition of a C. pecorum infection by a koala and that all C. pecorum strains now infecting koalas are descended from that founding strain is unlikely to be correct. The most plausible model is that koalas have obtained C. pecorum infections as a result of a series of cross-species transmission events, possibly from pigs and/or ruminants.