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Furoviruses are bipartite viruses causing mosaic symptoms and stunting in cereals. Infection with these viruses can lead to severe crop losses. The virus species Furovirus tritici with soil-borne wheat mosaic virus (SBWMV), Furovirus cerealis with soil-borne cereal mosaic virus (SBCMV) and Furovirus japonicum with Japanese soil-borne wheat mosaic virus (JSBWMV) and French barley mosaic virus (FBMV) as members are biologically and genetically closely related. Here, we develop SYBR green-based real-time quantitative RT-PCR assays to detect and quantify the RNA1 and RNA2 of the three virus species. Using experimental data in combination with Tm-value prediction and analysis of primer and amplicon sequences, we determine the capacity of our method to discriminate between the different viruses and evaluate its genericity to detect different isolates within the same virus species. We demonstrate that our method is suitable for discriminating between the different virus species and allows for the detection of different virus isolates. However, JSBWMV RNA1 primers may amplify SBWMV samples, bearing a risk for false positive detection with this primer. We also test the efficiency of polyclonal antibodies to detect the different viruses by ELISA and suggest that ELISA may be applied as a first screening to identify the virus. The real-time qRT-PCR assays developed provide the possibility to screen for quantitative disease resistance against SBCMV, SBWMV and JSBWMV. Moreover, with our method, we hope to promote research to unravel yet unresolved questions with respect to furovirus–host interaction concerning host range and resistance as well as regarding the role of multipartite genomes.