A simple and effective method was developed for characterization of phenolic constituents in the roots of Polygonum multiflorum by combination of ultra-high-pressure liquid chromatography with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap). Stilbenes, anthraquinones, tannins and naphthalenes were differentiated by diagnostic fragment ions with accurate mass measurements and characteristic fragmentation pathways. Based on the proposed strategy, fifty-nine constituents were characterized or tentatively identified, of which twenty-two constituents were the first to be reported in P. multiflorum and twelve compounds were characterized as potential new compounds. The identification and structure elucidation of these chemicals provided essential data for further phytochemical studies and quality control of P. multiflorum. The results also demonstrated that our novel method can be extended to screen and characterize other phenolic constituents and their metabolites in botanical extracts. (C) 2012 Elsevier B.V. All rights reserved.