Burkholderia pseudomallei is the causative agent of melioidosis and was classified as a biologic agent by the Centers for Disease Control and Prevention (Atlanta, GA). Acute melioidosis has a case fatality rate of >40%, and septicemia is fatal in up to 90%. The aim of the study was to design 5'-nuclease real-time PCR assays for the rapid and reliable identification of the B. mallei/B. pseudomallei complex. Real-time PCR assays using TaqMan probes targeting the 16S rDNA and fliC were developed on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Specificity was assessed with 64 B. pseudomallei, nine B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. Sensitivity, specificity, and positive and negative predictive value of the assays were 100%. Discrimination between B. pseudomallei and B. mallei, an organism which can be regarded as a clone of B. pseudomallei, could not be achieved. A probit analysis revealed that 7.5 and 52 genome equivalents (GE) of B. pseudomallei could be detected using the fliC and the 16S rDNA assays (P = .05), respectively. In spiked blood samples, the detection limit was approximately 300 and 3.000 GE for fliC and the 16S rDNA, respectively. In conclusion, we recommend the simultaneous use of the 16S rDNA and fliC real-time PCR assays for the rapid and specific identification of the B. mallei/B. pseudomallei complex in positive blood cultures or from suspicious bacterial colonies allowing the early onset of appropriate antibiotic therapy.