An efficient and sensitive profiling approach to complex yew samples was developed using ultra-performance liquid chromatography/electrospray ionization mass spectrometry (UPLC/ESI-MS). The UPLC-based method displayed short analytical time and improved peak capability, as well as high sensitivity. The appropriate in-source collision-induced dissociation (CID) energy was employed to produce informative characteristic ions which could be used for stereochemical and sub-structural assignment of yew constituents. The method was successfully applied in the rapid screening of yew hair roots from various species, and 53 constituents including 47 taxoids were detected from partially purified root extract. Notably, C-7 hydroxytaxane stereoisomers could be identified based on their different fragment ions under the optimal profiling conditions. It was also observed that hair roots from different Taxus species exhibited nearly identical chemical distribution, indicating they had similar metabolic frameworks. Additionally, Taxus root resources also display benign medicinal perspective because they have relatively simple chemical profiles and possess high yields of valuable taxanes such as paclitaxel, cephalomannine, 10-deacetylpaclitaxel and 7-xylosyltaxanes. Copyright © 2008 John Wiley & Sons, Ltd.