The level of similarity between the DNA of Lolium multiflorum and three Festuca species, F. arundinacea, F. pratensis and F. glaucescens, was analysed on Southern blots, using DNA–DNA hybridization, and also on chromosomes using genomic in situ hybridization (GISH). It was demonstrated that the close relationship between L. multiflorum and the allohexaploid F. arundinacea arises principally from the affinity of L. multiflorum to one of the ancestral progenitors of F. arundinacea, namely F. pratensis. Using probes made from total genomic DNA of L. multiflorum, and also of L. perenne, particular regions of high homology, described here as ‘GISH bands’ were identified on F. pratensis chromosomes. These GISH bands, in combination with a specific probe for rDNA, provide us with new markers for the identification of chromosomes within the F. pratensis complement. Restriction analysis within the rDNA repeat unit revealed additional information on the close phylogeny of L. multiflorum and F. pratensis. The rDNA restriction patterns confirm that F. arundinacea originated as a hybrid between F. pratensis and F. glaucescens.