To compare and establish the HPLC fingerprint of Xanthium sibiricum and Xanthium mongolicum fruits for the quality control.Sample was separated on C18 column (250 mm x 4.6 mm, 5 μm) with the mobile phase consisting of methanol-0.2% phosphoric acid solution by gradient elution. The flow rate was 1.0 mL/min and wavelength was set at 327 nm.HPLC fingerprints of Xanthium sibiricum and Xanthium mongolicum fruits were established, and 17 common peaks were found. The HPLC fingerprints of Xanthium sibiricum fruits from different habitats had good similarities with similaritis over 0.9, but some compositions' relative contents of Xanthium mongolicum fruits were different compared with Xanthium sibiricum.This method is accurate, reproducible and stable, which can be used for the quality control of Xanthium sibiricum fruits and distinguishing it with Xanthium mongolicum fruits effectively.