Electrophoretic karyotypes were obtained from intact sporidia and mycelia of Tilletia controversa and T. caries, and hybrid progeny were obtained by crossing these pathogens. The chromosomes typically ranged from approximately 850 to 4,490 kilobases (kb) for all strains, and they were variable in number with 19 or 20 for strains of T. controversa, 14-20 for T. caries, and from 19 to 22 for the hybrid progeny. The estimated genome size varied from 28 to 42 megabases (Mb) for these strains. Radiolabeled probes made of single copy DNA fragments and a heterologous actin gene identified four linkage groups among all strains that exhibited maximum chromosome length polymorphisms of 14% or less. The chromosomes carrying the rDNA genes, representing a fifth linkage group, exhibited length polymorphisms of approximately 40%. The actin gene and a rDNA probe hybridized with one or more bands in these strains, suggesting that some of the variability in chromosome number may result from aneuploidy. The karyotypes of the hybrid progeny revealed chromosome numbers and genome sizes essentially identical to each parental strain, clearly indicating that the reduction division stage of meiosis had occurred. These data and other corroborative genetic data provide substantial evidence that T. controversa and T. caries are not different species, but variants of a single species.