Twenty-six isolates representing six morphologically similar taxa of Tilletia occurring in the western U.S.A. were compared using randomly amplified polymorphic DNA (RAPD) markers and restriction fragment length polymorphisms (RFLP) of the 5·8 s rDNA and flanking ITS regions. Two distinct clusters were separated in the dendrogram based on the RAPD analysis. One cluster contained isolates of T. controversa, Apera interrupta-infecting isolates of a potentially new species of Tilletia, and isolates of the Bromus-infecting varieties bromi-tectorum and guyotiana of the T. fusca (=T. bromi) complex. The second cluster contained Vulpia-infecting isolates of T. fusca var. fusca. The separation of the Bromus-infecting varieties from the Vulpia-infecting variety of T. fusca was supported by distinct restriction digest phenotypes. The RAPD analysis was more sensitive than the RFLP analysis, and allowed separation of the Bromus-infecting varieties bromi-tectorum and guyotiana corresponding to host specificity shown in previous studies. The presence of genetically distinct populations of T. fusca var. fusca occurring on V. microstachys and V. octoflora is indicated by RAPD and RFLP analyses. Species status of T. togwatii, a bunt infecting Poa reflexa that was previously removed from the T. fusca complex, is also supported by RAPD and RFLP analyses.