ABSTRACT We present a duplex, real-time PCR assay for detection of Klebsiella pneumoniae carbapenemase ( bla KPC ) and New Delhi metallo-β-lactamase ( bla NDM ) genes. Accuracy was assessed with 158 Gram-negative bacillary isolates, including 134 carbapenemase producers. The assay had 100% sensitivity and specificity compared with reference methods and a turnaround time of 90 min.