Summary.To evaluate the quality of Fructus Arctii, an accurate and reliable method of high-performance liquid chromatography/diode array detection-electrospray ionization-mass spectrometry (HPLC/DAD-ESI-MS) was developed. Nine compounds, including chlorogenic acid, caffeic acid, trans-p-hydroxycinnamic acid, arctiin, arctignan A, ethyl caffeate, matairesinol, arctigenin, and lappaol B, were determined simultaneously in 19 batches of Fructus Arctii samples collected from different localities. Nineteen common peaks were identified or tentatively assigned by comparing their mass spectrometric data with reference compounds, self-established compound library, and published literatures. Also, the 19 common peaks were selected as characteristic peaks to assess the similarity of chromatographic fingerprinting of these samples. Moreover, hierarchical clustering analysis (HCA) and principal components analysis (PCA) were successfully applied to demonstrate the variability of samples. The results indicated the content of nine compounds that varied greatly among the samples, and 19 samples collected from different localities could be discriminated. Furthermore, chlorogenic acid, arctiin, and arctigenin were found to be chemical markers for evaluating the quality of Fructus Arctii.