Background: Penthorum chinense Pursh (P. chinense) is a well-known traditional Chinese medicine (TCM) plant, which has long been used for the prevention and treatment of hepatic diseases. This study aimed to genetically characterize the varieties of P. chinense from different geographic localities of China by random amplification of polymorphic DNA (RAPD)-PCR technique and verified with inter-simple sequence repeat (ISSR) markers.

Results: The P. chinense samples were collected from nine different geographic localities. Previously improved RAPD and ISSR markers were utilized for genetic analysis using DNA amplification. The genetic relationship dendrogram was obtained by conducting cluster analysis to the similarity coefficient of improved RAPD and ISSR markers. Improved RAPD yielded 185 scorable amplified products, of which 68.6% of the bands were polymorphic, with an average amplification of 9.25 bands per primer. The ISSR markers revealed 156 alleles with 7.8 bands per primers, where 59.7% bands were polymorphic. Furthermore, the similarity coefficient ranges of RAPD and ISSR markers were 0.71–0.91 and 0.66–0.89, respectively.

Conclusions: This study indicated that improved RAPD and ISSR methods are useful tools for evaluating the genetic diversity and characterizing P. chinense. Our findings can provide the theoretical basis for cultivar identification, standardization, and molecular-assisted breeding of P. chinense for medicinal use.