Background: Polygoni Multiflori Radix (PMR) is a widely distributed herb that has been used for centuries in the treatment of a range of systemic diseases among practitioners of traditional Chinese medicine. Objectives: The present comparative metabolite analysis study was conducted in an effort to understand the relationship between the geographical origin of PMR samples and their medicinal properties. Materials and Methods: As a metabolomics analysis of 35 PMR samples collected from Guangdong and other provinces in China was conducted via ultra-high performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry. Results: Differential metabolite profiles in these PMR samples were evaluated through multivariate statistical analyses. In total, this approach led to the identification of 778 differential metabolites [value of group contribution (VIP) >1, P < 0.01, and fold-change (FC) >2 or <0.5)] that were primarily associated with glycosaminoglycan degradation, lipoic acid metabolism, flavonoid biosynthesis, tyrosine metabolism, flavone and flavonol biosynthesis, phenylpropanoid biosynthesis, and phenylalanine metabolism. Of these metabolites, catechins accounted for seven significantly altered metabolites (VIP ≥2, P < 0.01, and FC >2 or <0.5) when comparing PMR samples from Guangdong with those from other regions. This suggests that PMR samples contain metabolite profiles characteristic of their provenance. Conclusion: As such, metabolomic profiling may be effective means of differentiating between PMR samples from different geographic regions within China, thus providing a sound theoretical basis for the reliable differentiation among and selection of pharmacologically optimal PMR samples.