Rising atmospheric CO2 concentrations and associated climate changes are thought to have contributed to the steady increase of Fusarium head blight (FHB) on wheat. However, our understanding of precisely how elevated CO2 influences the defense response of wheat against Fusarium graminearum remains limited. In this study, we evaluated the metabolic profiles of susceptible (Norm) and moderately resistant (Alsen) spring wheat in response to whole-head inoculation with two deoxynivalenol (DON)-producing F. graminearum isolates (DON+), isolates 9F1 and Gz3639, and a DON-deficient (DON-) isolate (Gzt40) at ambient (400 ppm) and elevated (800 ppm) CO2 concentrations. The effects of elevated CO2 were dependent on both the Fusarium strain and the wheat variety, but metabolic differences in the host can explain the observed changes in F. graminearum biomass and DON accumulation. The complexity of abiotic and biotic stress interactions makes it difficult to determine if the observed metabolic changes in wheat are a result of CO2-induced changes in the host, the pathogen, or a combination of both. However, the effects of elevated CO2 were not dependent on DON production. Finally, we identified several metabolic biomarkers for wheat that can reliably predict FHB resistance or susceptibility, even as atmospheric CO2 levels rise.