The Gram-negative bacterium Burkholderia pseudomallei (Bps) is a common environmental inhabitant in the Darwin region, Northern Territory. Bps causes melioidosis, a potentially deadly disease acquired from environmental exposure to the organism. In the past 24 years, ~820 cases have been reported in the Darwin region, yet tracing the origin of infection can be difficult due to its highly recombinogenic genome. One tool, multilocus sequence typing (MLST), has proven useful for tracing Bps origin. We have previously found that four Bps sequence types (STs) predominate in Darwin; STs 36, 109, 132 and 562. Despite its usefulness, MLST remains an expensive and laborious method. We therefore sought an inexpensive PCR-based alternative for identifying these common Darwin region Bps STs. Using MLST data, we were unable to identify ST-specific single-nucleotide polymorphisms (SNPs) due to the highly recombinogenic nature of Bps. We therefore interrogated our extensive WGS data of 289 mostly Australian Bps isolates, comprising 161STs. Phylogenetic analysis of 205,264 orthologous SNPs showed that isolates with identical STs grouped tightly together, confirming that MLST is a useful tool for ‘same’ or ‘different’ strain assignments in recombinogenic species. From the orthologous SNPs, 96, 52, 392 and 534 SNPs were 100% specific for STs 36, 109, 132 and 562, respectively. We subsequently designed inexpensive allele-specific real-time PCR assays for each of the four STs. The four assays were validated across 139 Australian isolates with known ST profiles; in all cases, SNPs were specific. Our findings are important for rapidly characterising Bps infections acquired from the Darwin region, and for source tracing in the improbable event that a Darwin region strain is bioweaponised. More broadly, our study demonstrates that accurate genetic targets can be identified in highly recombinogenic species, but highlights the importance of extensively-populated MLST and WGS datasets to counteract the effects of homoplasy.