Abstract: The study was undertaken to clarify the differences in metabolite groups between Aster yunnanensis and Pulicaria insignis to establish plant origin and identify plant resources. Non-target metabolomics of A. yunnanensis and P. insignis was undertaken with sample derivatives and gas chromatography-mass spectrometry (GC-MS). Principal component analysis (PCA), partial least-squares discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to characterize the differing metabolites of A. yunnanensis and P. insignis. Correlation analysis of these metabolites and metabolic pathway analysis allowed us to identify metabolic pathway changes between samples. Characterization of the antibacterial activity of the essential oil from A. yunnanensis and P. insignis showed that 384 metabolites were identified in the two species, including amino acids and peptides, phenylpropanoids, aromatics, glycoside, nucleotide, flavonoid, alkaloids, saccharides, vitamin, organic acid, lipids, esters, alcohol. A total of 92 differential metabolites with significant differences in content (P < 0.05, VIP > 1) were identified. In conclusion, a new method based on pre-column derivatives and GC-MS metabolomics was used to distinguish and compare A. yunnanensis and P. insignis metabolites.