Rhynchosporium consists of two species, R. secalis and R. orthosporum. Both are pathogens of grasses with R. secalis infecting a variety of Poaceae hosts and R. orthosporum infecting Dactylis glomerata. Phylogenetic analyses of multilocus DNA sequence data on R. secalis isolates originating from cultivated barley, rye, triticale and other grasses, including Agropyron spp., Bromus diandrus and Hordeum spp., resolved the monophyletic groups into three species according to their respective hosts. Host specificity according to phylogenetic lineages was confirmed with pathogenicity studies. Because R. secalis was described first on rye this name is retained for Rhynchosporium isolates infecting rye and triticale. Rhynchosporium isolates infecting cultivated barley and other Hordeum spp. and Bromus diandrus belong to a distinct species, R. commune. Similarly isolates infecting Agropyron spp. represent a distinct species of Rhynchosporium, namely R. agropyri. A PCR-RFLP assay was developed as a rapid tool for species identification of R. secalis and R. commune.