The rust species Puccinia graminis and P. striiformis sensu stricto are important cereal pathogens, most well-known for the potential of causing disease epidemics on wheat and generating significant yield losses. Early and accurate detection of the pathogens would facilitate effective control of the diseases. In the present study we developed real-time PCR assays to detect the specific lineages that include the wheat pathogens for each species complex, as identified using multi-gene DNA sequence analyses. Four DNA loci, for a comprehensive set of target and closely related fungi collected from diverse hosts and geographic regions, were explored to search for suitable lineage-specific probes: β-tubulin (BT), cytochrome c oxidase subunit 1 (COI), rDNA internal transcribed spacer (ITS), and RNA polymerase II second largest subunit (RPB2). Four TaqMan® real-time PCR assays were designed based on either the BT or RPB2 genes: one targeting Puccinia Series Striiformis (PSBT), one targeting P. striiformis sensu stricto (PSstrRPB2), and two targeting the P. graminis lineages on wheat (Pg2+BT and Pg2RPB2). Sensitivities of the assayswere determined to be 0.65 pg/uL (Pg2) and 5 pg/uL (PS). Specificity of each assay was confirmed using a broad diversity of rusts and selected other wheat-associated fungi. The ITS and COI loci were found to be unsuitable for diagnostic assay development but contributed phylogenetic signal to the multi-gene analyses.