The performance and portability of a multiplex real-time PCR assay to detect KPC, NDM, OXA-48-like and VIM carbapenemase gene families from bacterial isolates was assessed using Rotor-Gene Q and ABI 7500 instruments. Gram-negative bacterial isolates (n = 502) were comprised of 100 isolates each with KPC, NDM, VIM or OXA-48-like carbapenemases (including 17 with OXA-181) and 2 isolates with NDM + OXA-48-like enzymes (including 1 with OXA-181) as well as 100 assay-negative isolates comprised of 24 IMP-producers, 24 carbapenem-resistant isolates with no known carbapenemase gene and 52 extended-spectrum β-lactamase-producing carbapenem-susceptible isolates. A multicentre evaluation was carried out in five laboratories using a subset of 100 isolates comprised of 22 isolates each with KPC, NDM, OXA-48-like or VIM alleles and 12 isolates that were negative for the assay targets. Initial validation of the assay on both the Rotor-Gene Q and ABI 7500 instruments demonstrated 100% sensitivity amongst the 402 isolates that were positive for KPC, NDM, OXA-48-like (including OXA-181) and VIM carbapenemase genes, whilst the 100 assay-negative samples were correctly identified indicating 100% specificity. During the multicentre evaluation the same 100% level of sensitivity and specificity was observed in each of the five centres that participated. Neither invalid nor false-positive results were observed. In conclusion, the assay offers a portable and reliable option for the detection of bacteria carrying clinically significant carbapenemases encoded by KPC, NDM, VIM and OXA-48-like carbapenemase genes using either of the two most common real-time PCR instrument platforms.