Koala retrovirus (KoRV) is a gammaretrovirus that is becoming endogenous in koalas. Here, we explored the dynamics of KoRV infection in captive koalas in Japan. We isolated peripheral blood mononuclear cells (PBMCs) from 11 koalas, from which we extracted the KoRV genome. We found the prevalence of KoRV provirus in the koalas to be 100%, and the copy number of KoRV proviral DNA in genomic DNA isolated from PBMCs was variable. The KoRV envelope genes from 11 koalas were sequenced and all were found to be KoRV type A. Nucleotide substitution analysis revealed differences in the KoRV env gene sequences of parents and their offspring. Although no viral KoRV RNA was detected in plasma of healthy koalas, a high copy number was found in plasma of a diseased koala (#6). Hematological analysis showed a high white blood cell (WBC) count in the blood of koala #6. Notably, when retested ~ 5 months later, koala #6 was found to be negative for KoRV in plasma, and the WBC count was within the normal range. Therefore, KoRV in the plasma could be a possible indicator of koala health. We also investigated KoRV growth in concanavalin-A-stimulated koala PBMCs by measuring the KoRV provirus copy number in gDNA and the KoRV RNA copy number in cells and culture supernatants by real-time PCR at days 4, 7, and 14 post-culture. We also observed that KoRV isolates were able to infect HEK293T cells. These findings could enhance our understanding of the dynamics of KoRV and its pathogenesis in koalas.