Barley yellow mosaic virus (BaYMV) and Barley mild mosaic virus (BaMMV) are economically-important, soil-borne pathogens of winter barley. Until recently, laboratory diagnosis of these pathogens has relied upon ELISA using polyclonal antiserum. However, due to the unstable nature of these viruses, combined with the inability to sap transmit BaYMV, high quality antiserum has been difficult to obtain and as a result the performance of ELISA is often unsatisfactory. As an alternative approach two TaqMan assays (one BaYMV-specific and the other BaMMV-specific) have been developed. These assays have been validated for three seasons, by testing samples in parallel with ELISA. This testing indicates that TaqMan is a more reliable detection method than ELISA, especially with late-season and mixed infection samples. Data is also provided on a comparison of using simplex assays versus a multiplex assay for detecting BaYMV and BaMMV. The results indicate that while multiplexing does lead to a small reduction in sensitivity, it can be used reliably to streamline routine diagnosis. Further improvements in the development of a routine diagnostic procedure are also described including details of a rapid automated extraction procedure (Kingfisher) and the use of a novel cereal-specific control assay.