The Organization for Economic Co-operation and Development (OECD) recommends the measurement of specific plant components for compositional assessments of new biotechnology-derived crops. These components include proximates, nutrients, antinutrients, and certain crop-specific secondary metabolites. A considerable literature on the natural variability of these components in conventional and biotechnology-derived crops now exists. Yet the OECD consensus also suggests measurements of any metabolites that may be directly associated with a newly introduced trait. Therefore, steps have been initiated to assess natural variation in metabolites not typically included in the OECD consensus but which might reasonably be expected to be affected by new traits addressing, for example, nutritional enhancement or improved stress tolerance. The compositional study reported here extended across a diverse genetic range of maize hybrids derived from 48 inbreds crossed against two different testers. These were grown at three different, but geographically similar, locations in the United States. In addition to OECD analytes such as proximates, total amino acids and free fatty acids, the levels of free amino acids, sugars, organic acids, and selected stress metabolites in harvested grain were assessed. The major free amino acids identified were asparagine, aspartate, glutamate, and proline. The major sugars were sucrose, glucose, and fructose. The most predominant organic acid was citric acid, with only minor amounts of other organic acids detected. The impact of genetic background and location was assessed for all components. Overall, natural variation in free amino acids, sugars, and organic acids appeared to be markedly higher than that observed for the OECD analytes.