There are differences in the types and frequency of diseases between Queensland (QLD) and South Australian (SA) koala populations. It is hypothesised that Koala retrovirus (KoRV) contributes to this difference. All QLD koalas are KoRV-positive, and the proviral load is much higher when compared to SA koalas. These differences could potentially account for the variation in population disease. It is also possible that an association exists between KoRV env gene variants and disease. Currently 9 genetic variants (A-I) have been identified on the basis of hypervariable region diversity of env gene. To investigate an association between KoRV variants and disease, we compared KoRV env variant diversity in wild koala populations of QLD and SA. A high-throughput RNA-seq analysis was performed on RNA extracted from lymph node samples of 10 QLD and 19 SA koalas. We identified that KoRV-A is the most prevalent variant in koalas in both areas. The majority of QLD koalas had multiple other variants whereas only 1 of 17 KoRV positive SA koalas had KoRV-E in addition to KoRV-A. The genetic diversity of KoRV in QLD koalas may contribute to the higher levels of disease in this population compared to South Australian animals.