In the Darwin region of Australia where melioidosis is highly endemic, only 11/354 (3%) healthy residents were seropositive by indirect hemagglutination assay, despite extensive exposure to Burkholderia pseudomallei. None developed melioidosis, but some described a prior self-limiting illness. This seropositivity rate is much lower than that seen in northeast Thailand, where melioidosis is similarly highly endemic, potentially reflecting important differences between these two locations in the epidemiology of melioidosis.