Burkholderia pseudomallei is an environmental bacterium and the causative agent of melioidosis, a tropical infection highly endemic in northern Australia and Southeast Asia. While there is a considerable degree of genetic recombination and diversity amongst B. pseudomallei isolates and a robust global biogeographical structure, little is known about the population dynamics of the bacterium on a local scale. Expanding the current knowledge of B. pseudomallei distribution and population structure in the environment is important for future monitoring and better awareness of the disease. In this thesis, I surveyed soil and surface runoff from Darwin, northern Australia and Vientiane, Laos, two urban melioidosis-endemic settings to examine the distribution and local spread of B. pseudomallei. By combining whole-genome sequencing (WGS) comparative phylogenetics and Bayesian probability analysis, my results demonstrated that some sequence types (STs) are more widespread and established in the urban Darwin environment, while others are highly spatially clustered over a small geographic area. The high rate of sequence type (ST) diversity in B. pseudomallei isolates from drain sites also suggested that dissemination of B. pseudomallei in the environment may occur via surface runoff. This was further supported by findings in Laos, which also demonstrated that drains likely play a role in dispersal of B. pseudomallei in the environment, particularly during periods of heavy rainfall. Better awareness of the local B. pseudomallei population structure in Darwin also enabled the successful investigation of two melioidosis case clusters in captive zoo animals using WGS comparative analysis in conjunction with detailed pathology and epidemiological data. The knowledge gained from this thesis will allow for better understanding of B. pseudomallei phylogeography and source attribution and further supports the use of high-resolution WGS technology to enhance investigations into cases of melioidosis, which may help to develop future preventative public health measures throughout endemic areas.