Evaluating the quality of herbal medicines by morphological features is a convenient, quick, and practical method compared with other methods that mostly depend on modern instruments. Here, laser microdissection and ultra-performance liquid chromatography are combined with mass spectrometry to map the distribution of secondary metabolites in cells or tissues of a herb itself for correlating its bioactive components and morphological features. The root and rhizome of Rheum palmatum L. were taken as research target, which is the Chinese medicine, Radix et Rhizoma Rhei. According to fluorescent microscopic characteristics, 12 herbal cells or tissues of Radix et Rhizoma Rhei were separated by laser microdissection. Thirty-eight compounds were identified or tentatively characterized in the microdissected tissues. (+)-Catechin, 1-O-galloyl-2-O-cinnamoyl-β-D-glucose, and emodin were found to be the major components in most of the tissues. The brown ergastic substances found in rays of normal and anomalous vascular bundles as well as the parenchymatous cells of rhizome pith and the parenchymatous cells of root xylem contained higher than average amounts of these three components and more kinds of secondary metabolites. Overall, results suggest that Radix et Rhizoma Rhei of larger size and with conspicuous "brocaded patterns" and star spots are of higher quality as they tend to have greater contents of bioactive components. The study provides quantitative and specific criteria by which the quality of Radix et Rhizoma Rhei can be judged. This research also established a new, reliable, and practical method for direct profiling and imaging of secondary metabolites in any herbal tissue.