Rheum Species (Rhubarb) was widely known as a purgative and antiinflammatory agent. In the Chinese Pharmacopoeia, rhubarb was prescribed for three Rheum species, Rheum palmatum, R. tanguticum and R. officinale. Due to huge market demand, its fake or adulteration is frequently found in the market. To explore the cultivated germplasm resource of medicinal rhubarb and develop an ultimate identification method for plants and drugs, molecular identification of the chloroplast matK gene and rpl32 gene were performed on 41 samples from main planting area in northwest China. The results showed that the matK and rpl32 regions showed high success rate of PCR amplification and DNA sequencing, as well as superior discriminatory ability, and we found that there were mixed planting of different medicinal rhubarb in the Minle, Huzhu, Datong county and adulteration in the Longde county. The analysis of NJ trees indicated that the same species clustered together, the three medicinal Rheum species and adulteration can be distinguished by nucleotide differences. In future, we suggest that conservation and efficient management should be strengthened for cultivated germplasm resource of medicinal rhubarb, we should also pay more attention to super-barcoding or specific barcode that enhance ability to discriminate the closely related medicinal plants.