Thirty Chlamydia-free koalas, Phascolarctos cinereus, were moved from French Island National Park to three forests near Ballarat (Victoria). Chlamydial exposure and infection were monitored by antibody Enzyme-linked Immunosorbent Assay (ELISA), Direct Immunofluorescence (DIF) and Polymerase Chain Reaction (PCR) of swabs; its impact evaluated by clinical examination. Chlamydia was not detected on French Island. At the end of the study, 16 out of 17 koalas were Chlamydia antibody positive, and 11 out of 16 were also positive for the presence of Chlamydia in the uro-genital tract. C. pecorum infected seven out of nine koalas, one out of nine were infected by C. pecorum and C. pneumoniae and one out of nine by C. pneumoniae alone. This translocation trial shows a high incidence of infection of the translocated koalas, suggesting that the movement of Chlamydia-free animals to areas where the status of the disease is unknown, or the movement of infected animals to other sites where koalas are present, should not be considered as a management option without detailed pre-release research. Further studies should focus on ascertaining the longer term impact of the disease on individuals and population dynamic of this species.