Objectives To evaluate the AusDiagnostics MT CRE EU assay for the detection of carbapenemase and acquired colistin resistance genes in Gram-negative bacteria. Methods The assay allows the detection of bla KPC, bla OXA-48-like, bla NDM, bla VIM, bla IMP, bla SIM, bla GIM, bla SPM, bla FRI, bla IMI, bla GES (differentiating ESBL and carbapenemase variants), bla SME and mcr-1/-2. It was evaluated against a panel of isolates including Enterobacteriaceae, Pseudomonas spp. and Acinetobacter spp. retrospectively (n = 210) and prospectively (n = 182). Results The CRE EU assay was able to detect 268/268 carbapenemase genes, with 239 belonging to the 'big five' families (KPC, OXA-48-like, NDM, VIM and IMP) and 29 carbapenemase genes of the SIM, GIM, SPM, FRI, IMI, SME and GES families. It could distinguish between ESBL and carbapenemase variants of GES. It also allowed detection of mcr-1/-2 colistin resistance genes on their own or in isolates co-producing a carbapenemase. Conclusions The AusDiagnostics MT CRE EU assay offered wide coverage for detection of acquired carbapenemase genes. It required minimal hands-on time and delivered results in less than 4 h from bacterial culture.