Radix Bupleuri (Chaihu in Chinese) is a traditional Chinese medicine commonly used to treat colds and fevers. The root metabolome and transcriptome of two cultivars of B. chinense (BCYC and BCZC) and one of B. scorzonerifolium (BSHC) were determined and analyzed. Compared with BSHC, 135 and 194 differential metabolites were identified in BCYC and BCZC, respectively, which were mainly fatty acyls, organooxygen metabolites. A total of 163 differential metabolites were obtained between BCYC and BCZC, including phenolic acids and lipids. Compared with BSHC, 6557 and 5621 differential expression genes (DEGs) were found in BCYC and BSHC, respectively, which were annotated into biosynthesis of unsaturated fatty acid and fatty acid metabolism. A total of 4,880 DEGs existed between the two cultivars of B. chinense. The abundance of flavonoids in B. scorzonerifolium was higher than that of B. chinense, with the latter having higher saikosaponin A and saikosaponin D than the former. Pinobanksin was the most major flavonoid which differ between the two cultivars of B. chinense. The expression of chalcone synthase gene was dramatically differential, which had a positive correlation with the biosynthesis of pinobanksin. The present study laid a foundation for further research on biosynthesis of flavonoids and terpenoids of Bupleurum L.