Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes ( to , and variants) in was developed for surveillance or research purposes. We designed four new primer pairs to amplify , , and gene products and used the originally described primers for to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described genes and their variants present in . The protocol was validated testing 49 European and isolates of animal origin. Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect and as singletons or in different combinations as they were present in the test isolates. One new variant, , was also identified. This method allows rapid identification of -positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.