DREBs are transcription factors that regulate abiotic stress tolerance in plants. Previously, we reported that wheat transgenic lines overexpressing GmDREB1 showed increased tolerance to drought and salt stress. However, the molecular basis of increased tolerance is still poorly understood, and whether the overexpression of DREB will cause unexpected effects is also of concern. We performed seed metabolic profiling of the genetically modified (GM) wheat T349 and three non-GM cultivars with LC-MS to identify the metabolic basis of stress tolerance and to assess the unexpected effects of exogenous gene insertion. Although we did not note the appearance of novel metabolites or the disappearance of existing metabolites, overexpression of the transcription factor GmDREB1 in T349 wheat influenced metabolite levels in seeds. Increased levels of stress tolerance-associated metabolites were found in the stress-sensitive non-transgenic acceptor counterpart J19, while metabolites associated with cell membrane structure and stability accumulated in T349. Among these metabolites in T349, most showed levels similar to those in the non-GM wheats. Overexpression of GmDREB1 in T349 may cause a shift in its metabolic profile leading to down-regulation of several energy-consuming processes to favor increased yield under stress conditions, which is a reasonable expectation of breeders while creating the GM wheat and GmDREB1 overexpression did not cause unexpected effects in T349 seeds. These results may be helpful for GM crop research and risk assessment.