<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>Introduction</jats:title><jats:p>Metabolomics is an 'omics' approach that aims to comprehensively analyse all metabolites in a biological sample, and has great potential for directly elucidating plant metabolic processes. Increasing evidence supports the view that plants produce a broad range of low‐molecular‐weight secondary metabolites responsible for variation from species to species, thus enabling the use of secondary metabolite profiling in the chemotaxonomy.</jats:sec><jats:sec><jats:title>Objective</jats:title><jats:p>To gain deeper insights into the metabolites to increasing plant diversity, we performed systematic untargeted metabolite profiling to exploit the different parts and species of <jats:italic>Aconitum</jats:italic> as a case study.</jats:sec><jats:sec><jats:title>Method</jats:title><jats:p>Application of ultraperformance liquid chromatography–quadrupole time‐of‐flight–high‐definition mass spectrometry (UPLC–QTOF–HDMS) equipped with electrospray ionisation and coupled with pattern recognition analyses to study constituents in the root of two kinds of <jats:italic>Aconitum</jats:italic> species.</jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Twenty‐two metabolites between the mother root of <jats:italic>Aconitum carmichaelii</jats:italic> Debx (CHW) and lateral root of <jats:italic>Aconitum carmichaelii</jats:italic> Debx (SFZ) and 13 metabolites between the CHW and root of <jats:italic>Aconitum kusnezoffii</jats:italic> Reichb (CW) have been identified. Of note, songorine, carmichaeline and isotalatizidine did not exist in CW, whereas they are present in the SFZ and CHW.</jats:sec><jats:sec><jats:title>Conclusion</jats:title><jats:p>Metabolomics based UPLC–QTOF–HDMS with multivariate statistical models was effective for analysis of constituents in the root of two kinds of <jats:italic>Aconitum</jats:italic> species. Copyright © 2012 John Wiley & Sons, Ltd.</jats:sec>