Technical Abstract: Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TRIMV) are widespread throughout the southwestern Great Plains states. Using conventional diagnostics such as Enzyme-Linked Immunosorbent Assays (ELISA), these two viruses are commonly found together in infected wheat samples. Methods for molecular detection have been developed for wheat viral pathogens, but until recently no multiplex method for detection of both WSMV and TriMV within a single sample was available. Therefore, the objective of this study was to develop a multiplex real-time PCR technique for detection of both pathogens within a single plant sample. Specific primers and probe combinations were developed for detection of WSMV and TriMV, single and multiple reactions were run simultaneously to detect any loss in sensitivity during the multiplex reaction, as well as any cross reaction with other common wheat viruses. The multiplex reaction was successful in detection of both pathogens, with little difference between single and multiplex reactions, and no cross reaction was found with other common wheat viruses. This multiplex technique will be useful not only for diagnostic evaluations, but also as a valuable tool for ecological and epidemiology studies, and investigations of host/pathogen interactions, especially when the host is infected with both pathogens.