The Check-MDR Carba test (Check-Points, Wageningen, Netherlands), which is based on specific molecular recognition of blaNDM, blaKPC, blaOXA-48, blaVIM, and blaIMP genes by DNA probe ligation and real-time PCR detection, was evaluated on 183 well-characterized Gram-negative rods. Representatives of the 5 gene families were accurately identified (specificities and sensitivities of 100%) within 4.5 hours. This test may be helpful to differentiate carbapenem resistance mediated by carbapenemases from those involving other mechanisms.