A high-performance liquid chromatographic (HPLC) method with ultraviolet absorption detection was developed to determine the presence of anthraquinones, polydatin, and resveratrol in Polygonum multiflorum Thunb. as well as other medicinal Polygonum species, viz., P. cuspidatum, P. oriental, P. aviculare, and P. vulgare, as well as commercial products that claim to contain P. multiflorum. The best results were obtained with a Phenomenex Gemini C18 column using gradient mobile phase composed of water (0.1% acetic acid) and acetonitrile (0.1% acetic acid). Elution was at a flow rate of 1.0 mL/min. The detection wavelength was 280 nm for anthraquinones and 320 nm for polydatin and resveratrol. The main anthraquinones identified were emodin and physcion. The HPLC pattern of P. multiflorum was also compared with 5 other species of Polygonum. The method was validated for precision, repeatability, and accuracy. The relative standard deviation was between 0.9 and 1.6%. The method was sensitive, quick, and accurate for determination of anthraquinones, polydatin, and resveratrol in 6 different species of Polygonum and can be used for quality control of P. multiflorum. The commercial samples and the 6 Polygonum species were compared microscopically, and a detailed description is provided for P. multiflorum.