OBJECTIVES: The Australian Leishmania (Mundinia) macropodum parasite causes cutaneous leishmaniasis (CL) among marsupial species. While CL is a major public health burden globally, it is not clear if humans are naturally exposed to the unique L. macropodum. To assess whether humans have an IgG response to L. macropodum, we examined anti-Leishmania antibodies among humans residing in a region of marsupial Leishmania endemicity in Australia. METHODS: Employing a serological enzyme-linked immunosorbent assay, we characterized Leishmania-specific IgG and IgG subclass responses to soluble Leishmania antigen (SLA) from L. macropodum and other Leishmania species (L. donovani, L. major and L. mexicana) in 282 blood donor samples. RESULTS: We found 20.57% of individuals demonstrated a positive total IgG response to L. macropodum. For individuals with antibodies to SLA from one Leishmania species, there was no increased likelihood of recognition to other Leishmania species. For samples with detectable L. macropodum IgG, IgG1 and IgG2 were the prevalent subclasses detected. CONCLUSIONS: It is not yet clear whether the IgG antibody detection in this study reflects exposure to Leishmania parasites or a cross-reactive immune response that was induced against an unrelated immunogen. Future studies should investigate whether L. macropodum can result in a viable infection in humans.