Cholera is an important enteric disease, which is endemic to different regions of the world and has historically been the cause of severe pandemics. Vibrio cholerae is a natural inhabitant of the aquatic environment and the toxigenic strains are causative agents of potentially life-threatening diarrhoea. A multiplex, real-time detection assay was developed targeting four genes characteristic of potentially toxigenic strains of V. cholerae, encoding: repeat in toxin (rtxA), extracellular secretory protein (epsM), mannose-sensitive pili (mshA) and the toxin coregulated pilus (tcpA). The assay was developed on the Cepheid Smart Cycler using SYBR Green I for detection and the products were differentiated based on melting temperature (Tm) analysis. Validation of the assay was achieved by testing against a range of Vibrio and non-Vibrio species. The detection limit of the assay was determined to be 10(3) CFU using cells from pure culture. This assay was also successful at detecting V. cholerae directly from spiked environmental water samples in the order of 10(4) CFU, except from sea water which inhibited the assay. The incorporation of a simple DNA purification step prior to the addition to the PCR increased the sensitivity 10 fold to 10(3) CFU. This multiplex real-time PCR assay allows for a more reliable, rapid detection and identification of V. cholerae which is considerably faster than current conventional detection assays.