Willd. (HD) and (L.) Lam. (HC), two closely related species of the same genus, are both used for health benefits and disease prevention in China. HC is also indiscriminately sold as HD in the wholesale chain and food markets. This confusion has led to a growing concern about their identification and quality evaluation. In order to further understand the molecular diversification between them, we focus on the screening of chemical components and the analysis of non-targeted metabolites. In this study, UPLC-QTOF-MS, UNIFI platform and multivariate statistical analyses were used to profile them. Firstly, a total of 113 compounds, including 80 shared chemical constituents of the two plants, were identified from HC and HD by using the UNIFI platform. Secondly, the differences between two herbs were highlighted with the comparative analysis. As a result, a total of 33 robust biomarkers enabling the differentiation were discovered by using multivariate statistical analyses. For HC, there were 18 potential biomarkers (either the contents were much greater than in HD or being detected only in HC) including three iridoids, eight flavonoids, two tannins, two ketones, one alcohol and two monoterpenes. For HD, there were15 potential biomarkers (either the contents were much greater than in HC or being detected only in HD) including two iridoids, eight flavonoids, one tannin, one ketone, and three anthraquinones. With a comprehensive consideration of the contents or the MS responses of the chemical composition, Hedycoryside A and B, detected only in HC, could be used for rapid identification of HC. The compounds 1,3-dihydroxy-2-methylanthraquinone and 2-hydroxy-3-methylanthraquinone, detected only in HD, could be used for rapid identification of that plant. The systematic comparison of similarities and differences between two confusing Chinese herbs will provide reliable characterization profiles to clarify the pharmacological fundamental substances. HC should not be used as the substitute of HD.