Many ascomycete Fusarium spp. are plant pathogens that cause disease on both cereal and noncereal hosts. Infection of wheat ears by Fusarium graminearum and F. culmorum typically results in bleaching and a subsequent reduction in grain yield. Also, a large proportion of the harvested grain can be spoiled when the colonizing Fusarium mycelia produce trichothecene mycotoxins, such as deoxynivalenol (DON). In this study, we have explored the intracellular polar metabolome of Fusarium spp. in both toxin-producing and nonproducing conditions in vitro. Four Fusarium spp., including nine well-characterized wild-type field isolates now used routinely in laboratory experimentation, were explored. A metabolic “triple-fingerprint” was recorded using ¹H nuclear magnetic resonance and direct-injection electrospray ionization–mass spectroscopy in both positive- and negative-ionization modes. These combined metabolomic analyses revealed that this technique is sufficient to resolve different wild-type isolates and different growth conditions. Principal components analysis was able to resolve the four species explored—F. graminearum, F. culmorum, F. pseudograminearum, and F. venenatum—as well as individual isolate differences from the same species. The external nutritional environment was found to have a far greater influence on the metabolome than the genotype of the organism. Conserved responses to DON-inducing medium were evident and included increased abundance of key compatible solutes, such as glycerol and mannitol. In addition, the concentration of γ-aminobutyric acid was elevated, indicating that the cellular nitrogen status may be affected by growth on DON-inducing medium.