AbstractThe total alkaloids and its five monomeric alkaloids were studied from the stems of 14 species of the genus Berberis from China. The optimal extraction conditions of the total alkaloids were established as including a ratio of liquid‐to‐material of 249.55:1 (ml/g), extraction temperature of 59.6°C, and extraction time of 137.5 min. The contents of total alkaloids of the 14 Berberis species range from 7.74 to 29.37 mg/g, three species, B. pruinosa, B. wilsonae, B. verruculosa, possess higher contents between 21.19 and 29.37 mg/g. The contents of five monomeric alkaloids were analyzed by ultra‐high‐performance liquid chromatography, using methanol and 0.2% formic acid as the optimal mobile phase, jatrorrhizine, berberine, and palmatine are found in all the 14 species, and three species, B. pruinosa, B. wilsonae, B. verruculosa, possess the higher contents between 1.99 and 9.48 mg/g, while epiberberine is detected in eight species with low contents (0.06–0.07 mg/g), and coptisine is detected in just three species with low contents (0.05–0.11 mg/g). Only one species, B. taliensis, is found to contain all five monomeric alkaloids. The results may promote the development and utilization of the largely unexploited abundant wild medicinal plant resources of Berberis species in China.