Abstract:Apis cerana unifloral honeys from vitex (Vitex negundo var. heterophylla Rehd.) and osmanthus (Osmanthus yunnanensis) were used as controls to identify the metabolite composition and characteristic substances in blueberry (Vaccinium Spp.) and wild rose (Rosa multiflora Thunb.). The metabolites of 39 specialty honey samples were clustered and analyzed using liquid chromatography-mass spectrometry untargeted metabolomics. Significantly changed metabolites were identified and functionally annotated. The 817 metabolites detected were enriched in 42 modules (e.g., amino acid metabolism (14 pathways), digestive system (10 pathways)), involving a total of 364 pathways. There were 336 significantly changed metabolites between blueberry and vitex honeys, 165 between blueberry and osmanthus honeys, 167 between blueberry and wild rose honeys, 270 between wild rose and vitex honeys, and 247 between wild rose and osmanthus honeys. Phenylalanyl-threonine and 3-hydroxy-4-methoxycinnamic acid were characteristic substances of blueberry honey, whereas N1,N5,N10,N14-tetra-trans-p-coumaroylspermine, eupatorin, and prop-2-enoic acid were characteristic substances of wild rose honey. Additionally, wild rose honey contained high contents of indole and its derivatives and glycerophospholipids. We identified and confirmed unique characteristic substances in blueberry and wild rose honeys, offering valuable references for subsequent identification of honey authenticity and traceability identification.