Background: Nowadays, Radix Polygoni Multiflori (RPM, Heshouwu in Chinese) from different geographical origins were used in clinic. In order to characterize the chemical profiles of different geographical origins of RPM samples, ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC-QTOF/MS) combined with chemometrics (partial least squared discriminant analysis, PLS-DA) method was applied in the present study. Methods: The chromatography, chemical composition and MS information of RPM samples from 18 geographical origins were acquired and profiled by UPLC-QTOF/MS. The chemical markers contributing the differentiation of RPM samples were observed and characterized by supervised PLS-DA method of chemometrics. Results: The chemical composition differences of RPM samples derived from 18 different geographical origins were observed. Nine chemical markers were tentatively identified which could be used as specific chemical markers for the differentiation of geographical RPM samples. Conclusions: UPLC-QTOF/MS method coupled with chemometrics analysis has potential to be used for discriminating different geographical TCMs. Results will help to develop strategies for conservation and utilization of RPM samples.