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Bupleuri Radix is the dry root of certain species of the genus Bupleurum and is commonly used in traditional Chinese medicine. The increasing global demand for Bupleuri Radix cannot be fulfilled with wild populations only. Therefore, cultivated Bupleurum is now the main commercial source of this medicinal product. Different species of Bupleurum show different medicinal properties and clinical effects, making reliable authentication and assignment of correct botanical origin for medicinal species critical. However, accurate identification of the cultivated Bupleurum species is difficult due to dramatic morphological variations resulting from cultivation. In this study, we sampled 56 cultivated Bupleurum populations of six different morphotypes (Types A-F) from the main production areas of China, and 10 wild populations of four species were used as reference materials. Conventional DNA barcoding was conducted to identify cultivated Bupleurum species. Additionally, verification based on complete chloroplast genomes was performed and new chloroplast markers were developed and evaluated. The combination of these methods resulted in the successful identification of all cultivated Bupleurum individuals. Three chloroplast regions are recommended as additional barcodes for the genus: ycf4_cemA, psaJ_rpl33, and ndhE_ndhG. This is a reliable and promising strategy that can be applied to the authentication of natural products and the identification of other medicinal plant species with similar taxonomic problems. [ABSTRACT FROM AUTHOR]