To identify Stagonospora avenae genetically, 53 isolates from Europe and North America were assessed by RFLP analysis and internal transcribed spacer (ITS) sequence polymorphisms in comparison with isolates of S. nodorum, S. arenaria and Septoria tritici. Our results also provided insight on possible phylogenetic divergence within the species currently identified by characteristic conidial morphology as S. avenae. Isolates from Europe, Canada, Minnesota and North Dakota showed little genetic variation. In RFLP and ITS sequence analyses, S. avenae isolates from New York and ATCC 12277 (origin unknown) had a closer relationship with barley-biotype S. nodorum, and S. avenae f. sp. triticea isolates of ATCC 26370 and ATCC 26377 from Minnesota were closely associated with wheat-biotype S. nodorum. Enzyme restrictions of ITS PCR-amplified products could facilitate the identification of Stagonospora on cereals.