ABSTRACT A collection of 712 Fusarium graminearum sensu stricto (s.s.) strains, predominantly gathered between 1999 and 2000 from nine states within the United States, was examined for population structure and polymerase chain reaction-based trichothecene type. Most strains belonged to a cohesive genetic population characterized by a 15-acetyldeoxynivalenol (15ADON) trichothecene type. However, using a Bayesian model-based clustering method, we also identified genetically divergent groups of strains in some sampled locations of Minnesota and North Dakota. Strains of the major group of divergent populations were of a 3ADON trichothecene type and formed a distinct cluster with a collection of previously gathered strains from Italy, which displayed all three trichothecene types (15ADON, 3ADON, and nivalenol). The co-existence of genetically divergent populations of F. graminearum s.s. in the Upper Midwest allows for the rejection of the hypothesis that F. graminearum s.s. in the United States consists of a single population. These results also suggest that recombination has been insufficiently frequent in this homothallic (selfing) fungal species to homogenize the divergent populations observed in the Upper Midwest.