Abstract: In order to study the difference of synthesis and accumulation of metabolites in different medicinal parts of Eucommia ulmoides Oilv. (Eucommiae Cortex, Eucommiae Folium and Eucommiae Flos Male), LC-Triple TOF MS/MS method combined with multivariate statistical analysis was used to analyze the difference of chemical constituents in different medicinal parts of Eucommia ulmoides Oilv. Through the analysis of the multistage tandem mass spectrometry, the characteristic peaks were extracted with mass spectrometry data peak matching, peak alignment, noise filtering. Hierachical cluster analysis (HCA), principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for data processing. The constituents were identified according to MS accurate mass and MS/MS spectrometry fragmentation information, combined with the software of database search, comparison with reference standards and literature. The results showed that the chemical constituents in Eucommiae Cortex, Eucommiae Folium and Eucommiae Flos Male were clearly distinguished. Kinds of 23 differential chemical constituents are screened and indentified, including aucubin, deacetyl asperulosidic acid, guaiacyl glycerol, geniposidic acid, chlorogenic acid, cryptochlorogenic acid, geniposide, caffeic acid, pinoresinol-di-O-β-D-glucopyranoside, rutin, kaempferol-3-O-rutinoside, isoquercitrin, eucomoside B, pinoresinol-4’-O-β-D-glucopyranoside, quercetin-3-O-malonyl-β-D-glucoside, astragalin, isochlorogenic acid A, kaempferol-3-O-(6″-O-acetyl)-β-D-glucopyranoside, naringenin, kaempherol and three kinds of quercetin glycoside, which present different changing laws. The different constituents among Eucommiae Cortex, Eucommiae Folium and Eucommiae Flos Male can provide the material basis for revealing the property and efficacy of different medicinal parts of Eucommia ulmoides Oilv.