Koala (Phascolarctos cinereus), an iconic marsupial species, is facing many threats to its survival and koala retrovirus (KoRV) is thought to be a contributor to this ongoing decline. KoRV infection occurs with 100% prevalence in Queensland (QLD) but is reported to have a lower prevalence in South Australia (SA). KoRV infection is associated with neoplastic and immunosuppressive diseases in koalas. There is anecdotal evidence that the severity of population disease associated with KoRV infection differs between QLD and SA, however the source and relevance of these differences are unknown. We hypothesized that proviral DNA and transcribed viral RNA levels might be different between QLD and SA koala populations and might be responsible for differences in disease severity. For this study, 157 blood samples were collected from QLD (n=60) and SA (n=97) koalas and proviral level measured by qPCR. When possible, viral RNA levels in plasma were also measured. Surprisingly, we found proviral inserts in all koalas except one SA koala and many SA koalas were PCR-positive for some genes of entire KoRV genome. The mean provirus level was higher in QLD than in SA animals. Interestingly, all (60/60) QLD koalas but only 22/42 (52.4 %) SA koalas were KoRV RNA positive in plasma. Viremia copy number in SA koalas was lower than QLD koalas. This study provides further understanding of the complex nature of KoRV infection in different koala populations and hints that factors such as degree of provirus truncation, quantity of provirus and whether virus is expressed may contribute to pathogenesis and severity of disease.