A subtraction library of Burkholderia pseudomallei was constructed by subtractive hybridisation of B. pseudomallei genomic DNA with Burkholderia thailandensis genomic DNA. Two clones were found to have significant sequence similarity to insertion sequences which have previously not been found in B. pseudomallei (designated ISA and ISB); and two clones showed sequence similarity to different regions of Burkholderia cepacia IS407 that has recently been detected in B. pseudomallei. The former, though possibly non-functional, represents new transposable genetic elements of B. pseudomallei. All three sequences were found to be present in multi-copy in the genomes of a number of B. pseudomallei strains and in B. thailandensis, which are the first transposable elements identified in this species.