We describe an acute fatal human case of melioidosis acquired in Ipswich, a city at 27.5°S in southern Queensland, south of the area traditionally considered endemic for melioidosis in Australia. Molecular typing revealed that this patient isolate was genetically distinct from 2 other human and 1 bovine isolates of Burkholderia pseudomallei from the same region and from 4 tropical northern Australian strains. This finding suggests that if B. pseudomallei has been introduced to the region from northern Australia, it was not in recent times, and there has not been a point source of infection. Burkholderia pseudomallei is present in temperate southern Queensland, which hitherto has not been well appreciated. Clinicians should consider the diagnosis of acute melioidosis in patients with severe pneumonia or septicemia acquired in subtropical areas such as southern Queensland, particularly after heavy summer rains with flooding.