Isolates of Tilletia controversa and T. bromi were sampled from wheat and two Bromus species hosts, respectively, in the Pacific Northwest, and genetic variation within and among populations was determined. Fifty-one random amplified polymorphic DNA markers from eleven primers were treated as phenotypic 1 and 0 character state data to estimate similarities and analyze molecular variance (AMOVA) among populations and as putative genetic loci to carry out analyses of gene diversity. Phenotypic analysis of T. controversa and T. bromi isolates revealed two distinct clusters that were 37% similar. The T. bromi cluster was subdivided further into two groups, corresponding to host, with 40% similarity. Cluster analysis based on allele frequencies produced similar results and also supported two T. bromi groups based on host. No evidence of natural hybridization and introgression was detected between the T. controversa and T. bromi populations. Both AMOVA and gene diversity analyses detected moderate levels of differentiation among T. controversa populations, whereas T. bromi populations were highly differentiated. The level of genetic differentiation observed between the T. bromi populations on different Bromus species hosts supports the hypothesis that a high degree of host specificity exists in the wild grass-infecting smuts. We speculate that the higher level of genetic differentiation among the T. bromi populations compared with the T. controversa populations on wheat may be due to selection by a more genetically diverse host population.