Fusarium verticillioides is a major pathogen of corn and poses a significant risk to human health by producing mycotoxins that accumulate in kernels. Considerable efforts have focused on identifying genes involved in secondary metabolism and pathogenesis. The availability of a sequenced genome accelerates gene discovery and characterization, but functional genomics approaches are hindered when disruption of a gene results in a phenotype that is not readily distinguishable from the wild type. To address this problem, we developed a metabolomics approach to characterize gene function. The technique involves culturing two fungal strains (wild type and a mutant) under identical conditions, extracting as wide a range of metabolites as possible, analyzing the metabolomes by gas chromatography/mass spectrometry, and comparing the unique metabolic fingerprint of each strain.