To determine whether the three Siegesbeckia plants can all serve as the origins of HS, we compared their fingerprint chromatograms and inhibitory effects on inflammatory mediators. Chemical analyses showed that the three species have different profiles, although they have common peaks in their fingerprint chromatograms. Hierarchical cluster analysis (HCA) and principal component analysis (PCA) of the common peaks demonstrated that all samples of the three species tend to be species-dependently grouped and separated. Ten components contributing to the species discrimination were identified, of which 8 are long-chain fatty acids/esters, and 2 are darutoside and hythiemoside A. Inhibitory effects of the three species on NO production and IL-6 secretion in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages are different, with SG being the most and SP the least potent. These chemical and bioactivity assays support the notion that the three Siegesbeckia species cannot be equally used as the plant origins of HS.;This work provides a chemical and pharmacological basis for determining whether the three Siegesbeckia genus herbs SP, SO and SG can all serve as the origins of HS; and also provides pharmacological justifications for the clinical application of HS in treating inflammatory disorders.