Reynoutria multiflora roots are a classical herbal medicine with unique nourishing therapeutic effects. Anomalous vascular bundle (AVB) forming "cloudy brocade patterns" is a typical morphological feature of R. multiflora roots and has been empirically linked to its quality classification. However, scientific evidence, especially for AVB-specific specialised metabolites, has not been comprehensively revealed thus far. Herein, desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) analysis was applied to carry out an in situ analysis of specialised metabolites distributed specifically at the AVB and cork of R. multiflora roots. To enlarge the scope of compounds by DESI detection, various solvent systems including acetone, acetonitrile, methanol, and water were used to assist in the discoveries of 40 specialised metabolites with determined localization. A series of bioactive constituents including stilbenes, flavonoids, anthraquinones, alkaloids, and naphthalenes were found specifically around the brocade patterns. Notably, phospholipids were detected from R. multiflora roots by in situ analysis for the first time and were found mainly in the phloem of AVB (PAB). This is the first study to use gradient solvent systems in DESI-MSI analysis to locate the specialised metabolites distribution. The discovery of feature-specific compounds will bridge the empirical identification to precision quality control of R. multiflora roots. CI - Copyright (c) 2022 Elsevier Ltd. All rights reserved.