Thirty-five isolates of Gaeumannomyces graminis were tested for ability to infect wheat, rye and oats, and for DNA polymorphisms using nuclear ribosomal DNA (rDNA) RFLP patterns and RAPD analysis. In general, the cereal-attacking isolates could be readily assigned to the rye-attacking (R) or non-rye-attacking (N) subgroups of var. tritici, or to var. avenae, on the basis of either of these molecular approaches. A small number of isolates gave anomalous rDNA RFLP patterns, but could nevertheless be assigned to one of the three groups by RAPD analysis. Two related G. graminis var. tritici isolates (T1–1 and T1–2) clearly grouped with the N rather than the R var. tritici subgroup on the basis of molecular analysis but were pathogenic to rye, indicating that the ability to infect this host may have arisen more than once. An earlier phylogenetic study of Gaeumannomyces involving DNA sequence analysis of the internal transcribed spacers of the rDNA indicated that several oat-infecting isolates originally classified as G. graminis var. tritici could be grouped with var. avenae isolates. From the rDNA RFLP and RAPD analysis described here, however, these isolates appear to be intermediate between var. avenae and var. tritici, although it is not clear whether they represent evidence of inter-varietal sexual hybridization.