Abstract: In order to study the influence of the different origin on the synthesis and accumulation of metabolites in Polygoni Multifori Radix, the UPLC-Triple TOF MS/MS method combined with multivariate statistical analysis was used to analyze the differences of chemical compositions in Polygoni Multifori Radix from different habitats. Through the analysis of the multistage tandem mass spectrometry, the characteristic peaks were extracted with mass spectrometry data peak matching, peak alignment and noise filtering. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for data processing. The chemical compositions were identified or tentative presumed according to MS accurate mass and MS/MS spectrometry fragmentation information, combined with the software of database search and literature. The results showed that the differences among samples of Polygoni Multifori Radix from different habitats are distinguishable. A total of 36 differential chemical compositions were screened in Polygoni Multifori Radix from different habitats, and 33 chemical constituents of which were identified. Among of them, there were 12 kinds of common differential chemical compositions, such as 3,5-dihydroxyphenol-1-O-β-D-(6″-O-galloyl)-glucopyranoside, gallic acid, pocyanidin B-7-3 - O -gallate, catechin glycosidase, piceatannol-3-O-β-D-(6″-O-galloyl)-glucopyranoside, stilbene glucoside, THSG-glucuronide, torachrysone-O-β-D-glucopyranoside, emodin-8-O-(6′-O-malonyl)-β-D-glucopyranoside, emodin, palmidin A and physcion, which presented different change laws. The results showed that stilbene glucoside, torachrysone-O-β-D-glucopyranoside and emodin-8-O-(6′-O-malonyl)-β-D-glucopyranoside are much higher in the samples from Shibing county Guizhou province, 3,5-dihydroxyphenol-1-O-β-D-(6″-O-galloyl)-glucopyranoside, gallic acid and palmidin A are much higher in the samples from Yingshan city Hubei province, catechin glycosidase, piceatannol-3-O-β-D-(6″-O-galloyl)-glucopyranoside, THSG-glucuronide and emodin are much higher in the samples from Gaozhou city Guangdong province, pocyanidin B-7-3-O-gallate, and physcion are much higher in the samples from A’ba county Sichuan province. This study can provide basic information for revealing the influence law of the different origin on the biosynthesis of metabolites in Polygoni Multifori Radix.