Methods for the rapid detection and differentiation of the Burkholderia pseudomallei complex comprising B. pseudomallei, B. mallei, and B. thailandensis, have been the topic of recent research due to the high degree of phenotypic and genotypic similarities of these species. B. pseudomallei and B. mallei are the causative agents of melioidosis and glanders, respectively. B. pseudomallei and B. mallei are recognized by the CDC as tier 1 select agents. Although B. thailandensis is generally avirulent in mammals, this species displays very similar phenotypic characteristics to that of B. pseudomallei. Optimal identification of these species remains problematic, due to the difficulty in developing a sensitive, selective, and accurate assay. To date, no real-time, multiplex PCR assay has been developed that can detect and differentiate between B. pseudomallei, B. mallei, and B. thailandensis in a single tube format. Here, we describe the development of such an assay that detects and differentiates the species of the B. pseudomallei complex. A real-time quadruplex qPCR assay, Bcom, was designed to target unique genomic regions of B. pseudomallei, B. mallei, B. thailandensis, and the B. pseudomallei complex that detects and differentiates the three species. A total of 299 isolates within the B. pseudomallei complex was evaluated in this study, as well as 15 near-neighbors and other bacterial species. The results showed that this quadruplex assay was capable of detecting the respective species in a given sample at a sensitivity between 288 fg and 277 pg of genomic DNA. The B. pseudomallei- and B. pseudomallei complex-specific assays tested negative on two presumed B. pseudomallei isolates. In addition, a third presumed B. pseudomallei isolate tested negative by the B. pseudomallei-specific test, but was detected by the B. thailandensis and B. pseudomallei complex-specific assays. After cultural and biochemical characterization, 16s rRNA sequencing, and multiple loci sequencing, it is proposed that B. pseudomallei 34 is B. thailandensis 82172 (Accession No. DQ388536), B. pseudomallei Darwin 175 is Elizabethkingia meningoseptica, and B. pseudomallei 135 is a new strain of B. ubonensis 135.