An environmental surveillance programme was developed to determine whether water supplies could be a source of Burkholderia pseudomallei as noted during previous melioidosis outbreak investigations. Water supplies to communities in the three northern Australian jurisdictions (Western Australia, Northern Territory and Queensland) were sampled periodically during 2001 and 2002. Water and soil samples were collected from communities known to have had recent culture-positive melioidosis cases and nearby communities where no cases had been diagnosed. Clinical isolates of B. pseudomallei obtained from northern Australian patients during 2001 and 2002 were compared with the environmental B. pseudomallei isolates by ribotyping and pulsed-field gel electrophoresis. B. pseudomallei was isolated from 11 distinct locations, all in the Northern Territory, seven of which were associated with culture-positive melioidosis cases (>1 case at three locations). Water was implicated as a possible environmental source of melioidosis in six locations. A variety of free-living amoebae including Acanthamoeba and Hartmannella spp. that are potential hosts to B. pseudomallei were recovered from environmental specimens. Culturable B. pseudomallei was not found to be widely dispersed in the environments sampled.