As a primary characteristic of substantial equivalence, the evaluation of unintended effects of genetically modified plants has been evolving into an important field of research. In this study, a metabolic profiling method for rice seeds was developed using rapid resolution liquid chromatography/quadrupole time-of-flight mass spectrometry. The analytical properties of the method, including the linearity, reproducibility, intra-day precision and inter-day precision, were investigated and were found to be satisfactory. The method was then applied to investigate the differences between transgenic rice and its native counterparts, in addition to the differences found between native rice with different sowing dates or locations. Global metabolic phenotype differences were visualized, and metabolites from different discriminated groups were discovered using multivariate data analysis. The results indicated that environmental factors played a greater role than gene modification for most metabolites, including tryptophan, 9,10,13-trihydroxyoctadec-11-enoic acid, and lysophosphatidylethanolamine 16:0. The concentrations of phytosphingosine, palmitic acid, 5-hydroxy-2-octadenoic acid and three other unidentified metabolites varied slightly due to gene modification.