<sec> <title>Objectives</title> <p>Carbapenem resistance is a serious clinical and public health threat. Carbapenemase can confer carbapenem resistance, and most carbapenemase genes are plasmid encoded so resistance can easily spread. In this study, we aimed to develop a novel system based on the TaqMan platform for the rapid detection of 6 clinically prevalent carbapenemase genes: <italic>Klebsiella pneumoniae</italic> carbapenemase, New Delhi metallo-β-lactamase, oxacillinase, imipenem-hydrolyzing, Verona integron-encoded metallo-β-lactamase, and Guiana extended-spectrum β-lactamase.</p></sec> <sec> <title>Methods</title> <p>The triplex assay was verified by testing genomic DNA of 6 carbapenemase-producing <italic>Klebsiella pneumoniae</italic>. It was validated with a blinded panel of 310 Enterobacteriaceae isolates, including 225 carbapenemase-producers and 85 non-producers, by direct colony triplex real-time polymerase chain reaction (PCR). The real-time PCR was performed using the ABI 7500 fast instrument (Applied Biosystems, CA, USA) and specific primers for each carbapenemase target were designed to include modified peptide-nucleic acid oligonucleotides.</p></sec> <sec> <title>Results</title> <p>No amplification was detected among the negative samples. The result showed 100% concordance with the genotypes previously identified. The entire assay, including DNA extraction and real-time PCR, was completed within 2 hours.</p></sec> <sec> <title>Conclusion</title> <p>The newly developed triplex real-time PCR assay was useful for the rapid, accurate and simultaneous detection of 6 carbapenemase genes in Enterobacteriaceae, suggesting its potential to allow an early decision on the appropriate treatment, management, and prevention of the spread of resistant infections in hospitals.</p></sec>