Technical Abstract: Timely response to the threat of the wheat stem rust (Puccinia graminis f.sp. tritici; Pgt) race TTKSK and its variants (the 'Ug99' lineage) requires the availability of sensitive, fast and easily implemented diagnostic assays to accurately monitor movement of the pathogen by pathologists, breeders and diagnostic personnel. Existing molecular technologies such as DNA sequencing, SSR and AFLP marker assays are capable of discriminating between Ug99 and several races of Pgt, but none of these methods are suitable for rapid, high-throughput monitoring of Ug99, as they require either specialized equipment, a high level of user expertise, and/or require several days or weeks before results can be generated and analyzed. To overcome these limitations, we predicted that rapid and accurate identification of Ug99 could be routinely performed through real-time PCR technology, based on the detection of single nucleotide polymorphisms (SNPs) between Ug99 and other Pgt races. With the development of suitable SNP markers for use as DNA probes and optimized protocols, real-time PCR could provide a platform that could be implemented quickly and routinely in monitoring facilities, with only a minimum of user training, and would make use of equipment already available to most U.S. diagnostic and research laboratories. Homozygous SNPs for use in the development of real-time PCR hydrolysis probes were identified by mapping Illumina sequence data from four members of the Ug99 lineage as well as three additional Pgt isolates against the assembled reference genome. The Ug99 strains of the fungus represent the four variants of the pathogen that have been identified from Africa, with each member possessing distinct virulence profiles based on their response to stem rust resistance genes Sr24, Sr31 and Sr36. Thirty-eight probes and primer sets were selectively designed from the resultant database of >1 million SNPs, with each probe containing at least two SNPs, and primer sequences containing between 0-5 SNPs. When used to screen a worldwide collection of Pgt isolates, the probes were individually not capable of uniquely discriminating between Ug99 and other races of the fungus. However, when a suite of probes was used in combination, a fingerprint distinct for Ug99 was obtained in a repeatable manner. Our data show that once optimized, rapid and accurate SNP-based real-time PCR detection of Ug99 will be an important component in the identification and monitoring of this pathogen.