Lonicerae japonicae flos (LJF) and Lonicerae japonicae caulis (LJC) are derived from different parts of Lonicera japonica Thunb. (Caprifoliaceae), and have been used as herbal remedies to treat various diseases for thousands of years with confirmed curative effects. However, little attention has been paid to illustrating the differences in efficacy from the perspective of phytochemistry. In the present study, a simultaneous determination of 47 bioactive constituents, including 12 organic acids, 12 flavonoids, six iridoids, 13 amino acids and four nucleosides in 44 batches of LJF and LJC samples from different habitats and commercial herbs was established based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS). Moreover, principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and t-test were then performed to classify and reveal the differential compositions of LJF and LJC according to the content of the tested constituents. The results demonstrated that the types and contents of chemical components (e.g., isochlorogenic acid A, chlorogenic acid, neochlorogenic acid, quinic acid, secologanic acid, luteoloside, loganin, secoxyloganin, morroniside and L-isoleucine) were significantly different, which may lead to the classification and the differences in efficacy of LJF and LJC. Our findings not only provide a basis for the comprehensive evaluation and intrinsic quality control of LJF and LJC, but also pave the way for discovering the material basis contributing to the different properties and efficacies of the two medicinal materials at the phytochemical level.