Species delimitation in Taxus has been controversial and it is very difficult to distinguish yew materials by their morphological characters. In this paper, a valid HPLC fingerprinting method coupled with multivariate analysis was used to define a framework for Taxus species identification and classification. Fingerprint-based similarity was employed for a chemotaxonomic study by hierarchical clustering analysis (HCA) and principal component analysis (PCA). Based on the PCA loadings, twelve chemical constituents were selected as chemotaxonomic markers which can be used to establish a more practical classification. Finally, eight studied species could be divided into six well-supported groups and most samples can be assigned to the correct species. Additionally, twelve markers were tentatively identified by LC/MS.