[Show abstract][Hide abstract]ABSTRACT: Herba Oldenlandiae is the dried whole herb of Oldenlandia diffusa (Willd.) Roxb. (Family Rubiaceae) recorded in the Chinese pharmacopoeia. However, the herbs of O. corymbosa (L.) Lam and O. tenelliflora Bl. are also often available and used indiscriminately as Herba Oldenlandiae in the markets. In light of this, high-performance liquid chromatography (HPLC) combined with high resolution mass spectrometry (HR-MS) has been developed for a comparative analysis of chemical components in the three Oldenlandia species. The correlation coefficients of the entire chromatographic patterns of samples were calculated by software and used for appraising the similarity. The chemical information on the peaks in the chromatograms of O. diffusa was obtained by comparison of their exact mass data, UV spectra and literature values. The results indicated that the chemical profiling of O. diffusa is quite different in samples collected from different habitats. Furthermore, the variations in the abundance of asperuloside, E-6-O- P-coumaroylscandoside methyl ester and E-6-O- P-coumaroylscandoside methyl ester-10-methyl ether were significant in these related herbs. In conclusion, it is important to use the above three compounds as chemical markers for quality evaluation and chemical authentication of Herba Oldenlandiae and its substitutes.