Burkholderia pseudomallei is a soil-dwelling saprophyte and the cause of melioidosis. Multilocus sequence typing (MLST) of 266 Thai B. pseudomallei isolates (83 soil and 183 invasive) defined 123 sequence types (STs). Invasive isolates were over-represented in the 10 largest clones, and there was a significant difference in the classification index between environmental and disease isolates, confirming that genotypes were not distributed randomly between the two samples. MLST profiles for 158 predominantly invasive isolates from northern Australia contained a similar number of STs (96) as the Thai invasive isolates, but no ST was found in both populations. This analysis revealed strong genetic differentiation on the basis of geographical isolation, and a significant differentiation on the basis of virulence potential. The presence and distribution of five genomic islands described for B. pseudomallei K96243 was investigated in natural populations. In silico analysis of 10 B. pseudomallei genome sequences demonstrated variable presence of these regions, together with evidence for micro-evolutionary changes that generates GI diversity. Their presence was assessed in environmental (83) and invasive (103) B. pseudomallei isolates using PCR. Positivity ranged from 12% for a prophage-like island (GI 9), to 76% for a metabolic island (GI 16). The presence of each of the five GIs did not differ between environmental and disease-associated isolates. There was no reproducible association between the individual or cumulative presence of five GIs and clinical features in 103 patients with melioidosis. Burkholderia thailandensis is a non-pathogenic soil saprophyte which is highly related to B. pseudomallei. MLST of a collection of B. thailandensis isolates demonstrated two clusters. One cluster contained three isolates (one each from France, the U.S. and Cambodia), and the second larger cluster contained isolates from Thailand, Vietnam and Laos. eBURST analysis indicated that the larger cluster formed a single clonal complex, while all three strains of the small cluster were singletons.