Ziziphi Spinosae Semen (ZSS) as a traditional edible herb has been used for treatment of insomnia in China for centuries and developed as an additive for supplementary health food nowadays. Due to the scarcity of wild resources of ZSS, Ziziphi Mauritianae Semen (ZMS) as a folk medicine is widely used as products by substituting ZSS on the market. The current study aims to propose and verify a strategy being able to find the reliable chemical markers between ZSS and ZMS by the serial performances of 1H NMR, GC-MS and UHPLC-Q-Orbitrap-MS-based untargeted metabolomics. A total of 30 primary metabolites, 28 fatty acid and 83 secondary metabolites were detected and tentatively annotated in ZSS and ZMS. Significant species differences occurred amongst their chemome patterns. The most relevant classes of compounds from two seeds were genkwanin-C-glycoside, triterpene saponin, cyclopeptide alkaloid, statured fatty acids and polyunsaturated fatty acids, as well as amino acid and sugar. Finally, the distribution of 19 secondary metabolites and saccharose were able to highlight the species differences through receiver operating characteristics (ROC) curve assessment. The findings enable not only to present valuable chemical evidences for their discriminative quality criteria, but more importantly, to offer a robust tool being able to afford reliable markers through comparative metabolomics. © 2020 Elsevier Inc.