Less prevalent viruses of family Poaceae are usually excluded from the focus of interest, even though they represent a possible threat to agricultural production. We designed and validated a set of primer pairs suitable for detection and quantification of five RNA viruses, Lolium latent virus (LoLV), Oat necrosis mottle virus (ONMV), Ryegrass mosaic virus (RgMV), Soil-borne cereal mosaic virus (SBCMV), and Spartina mottle virus (SpMV), by means of one-step RT-qPCR based on SYBR Green I. These primers were used together with primers for Brome mosaic virus (BMV) and Wheat streak mosaic virus (WSMV) described elsewhere to screen grass and cereal samples from the Czech Republic. The results revealed a high prevalence of WSMV and RgMV, which pointed to possible local epidemics. We also make the first report of LoLV presence in the Czech Republic.