Biochemical differences between the wheat, rye and couch‐grass pathotypes of the eyespot pathogen, Pseudocercosporella herpotrichoides, have been identified by comparing isoenzyme profiles and DNA markers from several isolates. The wheat (W) and rye (R) pathotypes were clearly differentiated by both techniques, with isoenzyme profiling also separating the couch‐grass isolates from the W‐ and R‐type isolates. The isoenzyme profiles separated the P. herpotrichoides pathotypes from the two related species, P. anguioides and P. aestiva. The isoenzyme profiles of P. anguioides were closer to those of the R‐type isolates of P. herpotrichoides than the W‐type isolates, whereas the banding pattern of P. aestiva was very different. The isoenzyme profiles of the two German isolates, originally obtained from Nirenberg's laboratory, P. herpotrichoides var. herpotrichoides and var. acuformis, were similar to those of the wheat and rye pathotypes, respectively, isolated from UK‐infected material.