The prevalence of Chlamydia pecorum and Chlamydia pneumoniae infections in two free-range koala populations was assessed using genus-specific PCR combined with species-specific DNA probe hybridisation. Population A had a very high overall level of chlamydial infection (85%) with significantly more of these infections being due to C. pecorum (73%) compared to C. pneumoniae (24%). The second population had a much lower prevalence of infection (10%) with equal levels of both species. An important finding of this study was that, while five of 24 C. pecorum-infected koalas had clinical signs of the disease (both ocular and urogenital sites), none out of seven C. pneumoniae-infected koalas had signs of clinical disease. This suggests that C. pecorum may be the more pathogenic of the two chlamydial species infecting this host. The level of infection (assessed by intensity of the specific hybridisation signal) also differed between chlamydial species, with C. pecorum infections ranging from low to high grade whereas C. pneumoniae infections were always low grade. When the age of infected koalas was examined, 58% of young, sexually immature koalas were found to have C. pecorum infections, increasing to 100% of koalas in the older age groups. This suggests that, in this population at least, young koalas are readily infected with C. pecorum from their mothers. While the infection levels with C. pneumoniae were too low to be statistically significant, again, sexually immature koalas were found to be infected. The recent separation of chlamydial infections in koalas into two species is beginning to indicate different epizootiologies for koala C. pecorum compared to koala C. pneumoniae.