This study was designed to elucidate the biological variation in expression of many metabolites due to environment, genotype, or both, and to investigate the potential utility of metabolomics to supplement compositional analysis for substantial equivalence assessments of genetically modified (GM) crops. A total of 654 grain and 695 forage samples from 50 genetically diverse non-GM DuPont Pioneer maize hybrids grown at six locations in the U.S. and Canada were analyzed by coupled gas chromatography time-of-flight-mass spectrometry (GC/TOF-MS). A total of 156 and 185 metabolites were measured in grain and forage samples, respectively. Univariate and multivariate statistical analyses were employed extensively to compare and correlate the metabolite profiles. We show that the environment had far more impact on the forage metabolome compared to the grain metabolome, and the environment affected up to 50% of the metabolites compared to less than 2% by the genetic background. The findings from this study demonstrate that the combination of GC/TOF-MS metabolomics and comprehensive multivariate statistical analysis is a powerful approach to identify the sources of natural variation contributed by the environment and genotype.