An optimized HPLC-DAD method aided by similarity and hierarchical clustering analysis (HCA) was applied for identification of four species of the roots of Aconitum. The unique properties of this HPLC fingerprints and HCA were validated by analyzing Aconitum kusnezoffii Rchb. (AKR) samples and related herbs including A. karacolicum Rapcs. (AKP) samples, A. austroyunnanense W.T.Wang (AAW) samples and A. contortum Finet & Gagnepain (ACF). The results revealed that the chromatographic fingerprint combining similarity measurement and hierarchical cluster analysis could efficiently identify and distinguish AKR samples from its related species.