ABSTRACTBackgroundUncariae Ramulus cum Uncis (URCU) is a widely used traditional Chinese medicine (TCM) for treating neurodegenerative disorders. The Chinese Pharmacopeia recognizes five Uncaria species as authorized botanical sources of URCU; however, there is currently no established quality control method for individual species. Furthermore, significant variations in the chemical profiles among these species have been reported.ObjectiveChemical analysis methods were established to evaluate the five species in terms of chemical profile and resource availability, as to determine which species should be preferred as the official source(s).MethodTen batches of URCU sample were collected from various regions of China for each of the five species of URCU. Among five species, only UR can be obtained from herbal medicine market, whereas the other four species were sourced from the wild. All samples identities were confirmed by herbarium specimen authentication, morphological identification, and microscopic identification. Twelve alkaloids were identified in the five species, and a UPLC‐DAD assay method targeting six marker alkaloids was developed and validated.ResultsOnly UR and UM demonstrated the most stable chemical profiles and agreement with published literature. UR mainly contains corynoxeine, isocorynoxeine, rhynchophylline, and isorhynchophylline, whereas corynoxine, corynoxine B, rhynchophylline, and isorhynchophylline dominate in UM.ConclusionThis study comprehensively evaluated the chemical variations among the five officially recognized URCU species using a UPLC‐DAD method. It brings important implications for quality control and standardization of URCU. Considering chemical stability and supply availability, UR is recommended as the sole botanical source for URCU.