Abstract: As a traditional medicinal herb in China, Penthorum chinense Push from Gulin county is regarded as genuine regional drug with better clinical effects. To discriminate the geographical origin of six P. chinense samples cultivated in three provinces, Randomly Amplified Polymorphic DNA (RAPD) analysis was carried out with an improved method to increase the resolution and production using 10 mer-random primers. Similarity index was ranged from 0.61 to 0.97, which demonstrated that samples from different localities displayed similar band patterns. However, based on the analysis of selected 13 primers, primers SBS-I9, SBS-I20 and SBS-Q9 produced distinguishable bands among Sichuan, Yunnan and Hubei P. chinense. T-test of mean S.I. values of six accessions demonstrated the significant differences between Luzhou or Sichuan samples and others. Cluster analysis indicated that cultivars with close geographic distributions were clustered together consequently. This suggested that there are RAPD site variations and local specialized genotypes among the six samples. The results indicated that RAPD analysis is effective in distinguishing the geographical origin of genuine regional drug P. chinense grown in Luhzou, Sichuan. The approach is a valuable tool to authenticate other morphologically similar herbal medicinal materials.