Melioidosis is an infectious disease caused by Burkholderia pseudomallei. Genomic subtractive hybridisation was performed with the closely related avirulent species B. thailandensis to identify virulence genes of B. pseudomallei. The subtractive hybridisation products were highly specific for B. pseudomallei. Sequence analysis revealed a number of putative virulence factors, as well as apparently novel sequences of unknown function. The subtracted library contained DNA regions of relatively low G + C mol% content, which were distributed throughout the B. pseudomallei genome. The distribution of subtracted sequences amongst a collection of 22 B. pseudomallei isolates was found to be variable, with the exception of three strains which almost universally lacked the subtracted sequences. These three strains also differed in that they were highly haemolytic, indicating a possible separate virotype.