A new molecular test has been developed to help in the surveillance of Karnal bunt, a disease which produces bunted grains with rotting fish smell. Karnal bunt is caused by Tilletia indica and is the subject of strict quarantine regulations by many wheat growing countries including Australia. Any incursion would cause severe disruption to Australia’s international wheat trade and consequent huge losses in export markets. The protocol involves the release of DNA from spores, enrichment of DNA from Tilletia species and a final five-plex real-time quantitative PCR assay to detect, identify and distinguish T. indica and other commonly occurring Tilletia species (T. walkeri, T. ehrhartae, T. horrida, and a Tilletia group comprising T. caries, T. trabutii and T. fusca) in wheat grains. This fluorescent molecular tool has a detection sensitivity of 5-10 spores and thus bypasses the germination step in the current quarantine protocol required for confirmation when only a few spores have been found. The single–tube five-plex realtime assay contains five dual-labelled species-specific probes and associated species-specific primer pairs for the five target Tilletia species. The five different amplification products are quantitated and identified simultaneously by five different fluorescence spectra in a real-time instrument with at least five channels (Rotorgene 6000, Corbett Research, Australia). This protocol with its increased level of detection sensitivity and the technology to multiplex five fluorescent real-time PCR assay in one tube will greatly reduce workload and reagent costs and enables the implementation of an economically sustainable and effective Karnal bunt surveillance program.