Locational variations in the quantity of five hydroxyanthraquinone derivatives (emodin glycoside (1), chrysophanol glycoside (2), emodin (3), chrysophanol (4) and physcion (5)) in the rhizomes of Rheum emodi are described. A simple and reliable method was developed for quantitation of compounds (1–5) in the methanolic extract of rhizomes of R. emodi using reverse-phase high-performance liquid chromatography (HPLC) with photo-diode array detector (PDA). The separation was carried out using a Purospher ® -Star RP-18e column (4.6mm i.d.×250mm, 5μm) under the following conditions: acetonitrile:methanol (95:5, v/v) (solvent A) and water:acetic acid (99.9:0.1, v/v) (solvent B) as mobile phase with a linear gradient elution at a flow rate of 0.8mL/min. The detection wavelength was set at 290nm. Regression equation revealed a linear relationship (r 2 >0.9901) between the mass of hydroxyanthraquinone derivatives injected and the peak areas. The detection limits (S/N=3) ranged from 0.56 to 3.50ng/mL and the recoveries ranged from 95.7 to 103.5% for five hydroxyanthraquinone derivatives. Compound 2 was found in maximum quantity (up to 2.23%) in the rhizomes from all the three locations (L 1 , L 2 and L 3 ) while compound 5 was found in the least quantity (up to 0.19%).