A new high-performance liquid chromatographic (HPLC) fingerprinting method was developed for the quality control of Ganoderma lucidum. Twenty-nine batches obtained from three different origins in China were used to establish the fingerprint. The constituents of these samples were separated with a Kromasil C(18) column (250 mm x 4.6 mm, 5 microm) by linear gradient elution using water-acetic acid (100:0.1, v/v) and acetonitrile as mobile phase components at a flow rate of 0.8 ml/min and detector wavelength at 254 nm. Mean chromatograms and correlation coefficients of samples were calculated by the software "Similarity Evaluation System for Chromatographic Fingerprint of TCM". There were 19 common peaks in this fingerprint. Eleven of these common peaks were tentatively identified with reference to literature data based on their liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS) and UV data. This profile was then successfully used to identify and assess the differences among samples from various origins with the aid of similarity analysis. The diverse similarities among different samples indicated that the quality of G. lucidum was not stable and the products from different areas were inconsistent. All results showed that the developed fingerprint assay was specific and could further serve for quality identification and comprehensive evaluation of G. lucidum.