Rust diseases cause significant damage in forage and seed crops of perennial ryegrass (Lolium perenne), which is highly heterozygous and heterogeneous and thus presents difficulty in genetic analysis. There has been no definitive demonstration of the existence of pathotypes for stem rust or other rusts of perennial ryegrass, although experiments with crown rust (Puccinia coronata) of this host are strongly suggestive of pathotype specificity. We made single-pustule isolates of P. graminis subsp. graminicola, and applied them individually to a set of genetically diverse, clonally propagated individuals of L. perenne. There were clear examples of different patterns of virulence among isolates across the different plant clones, including qualitative and quantitative differences in resistance. These data demonstrate the existence of pathotype specificity in stem rust of L. perenne, information which will be useful in breeding for disease resistance.