Trichothecene mycotoxins are toxic metabolites produced by fungal pathogens, such as Fusarium graminearum (Fg), that infect cereal crops reducing grain quality and safety. The severity of trichothecene contamination is strongly dependent on the environment, and meteorological factors associated with climate change are predicted to increase the frequency and severity of conditions that favor disease outbreaks and contamination. However, it is unclear if the impact of meteorological factors directly affects Fg aggressiveness or indirectly affects disease outbreaks through stress induced changes in host metabolism. Rising atmospheric carbon dioxide (CO2) directly influences grain chemistry - particularly protein and carbohydrate content - which can indirectly impact mycotoxin production. Recently, we demonstrated that certain Fg strains produced more trichothecenes, on wheat grown at elevated CO2. To elucidate the direct effects of elevated CO2 on Fg trichothecene production, we inoculated grain of two barley varieties and two wheat varieties of different protein content with 15 Fg strains (5 strains from each North American population (NA1, NA2 and NA3)). The inoculated grain was placed into dark chambers controlled at 20°C, 50% relative humidity, and 400 ppm (ambient) or 1000 ppm (elevated) CO2. The fungus was allowed to grow and colonize the grain for 14 days, at which point the samples were collected for quantification of fungal biomass and trichothecene contamination. Results revealed that only fungal growth on wheat was affected by elevated CO2. Variety and Fg population and the interaction between the two significantly contributed to differences in trichothecene accumulation. Both wheat and barley varieties with relatively lower protein content accumulated more trichothecenes, and grains inoculated with NA3 population strains accumulated significantly higher amounts of trichothecenes in comparison to other populations.