Fusarium graminearum (Fg) is the primary fungal pathogen responsible for Fusarium head blight (FHB), a devastating disease of wheat and barley worldwide. FHB reduces crop yield and contaminates grain with trichothecene mycotoxins that are harmful to plant, human and animal health. Three genetically distinct populations of Fg, each associated with a different trichothecene chemotype, have been identified in North America (NA1, NA2, and NA3). To determine how this population-level diversity influences pathogenesis and mycotoxin contamination, we inoculated moderately resistant hard red spring wheat variety Alsen with 15 representative strains from each NA population and evaluated disease progression, mycotoxin accumulation, and fungal biomass. Additionally, we evaluated Fg population-specific differences in induced host defense responses. Population-specific differences in aggressiveness were observed and appear to be only partially attributable to differences in trichothecene chemotype. Differences in host defense response and in the relationship between trichothecene accumulation and fungal biomass were also observed between pathogen populations.