Introduction:Taxusspecies are used as medicinal plants all over the world. The leaves ofTaxusspecies are sustainable medicinal resources that are rich in taxoids and flavonoids. However, traditional identification methods cannot effectively identifyTaxusspecies on the basis of leaces used as raw medicinal materials, because their appearance and morphological characteristics are almost the same, and the probability of error identification increases in accordance with the subjective consciousness of the experimenter. Moreover, although the leaves of differentTaxusspecies have been widely used, their chemical components are similar and lack systematic comparative research. Such a situation is challenging for quality assessment.Materials and methods:In this study, ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry combined with chemometrics was applied for the simultaneous determination of eight taxoids, four flavanols, five flavonols, two dihydroflavones, and five biflavones in the leaves of sixTaxusspecies, namely,T. mairei,T. chinensis,T. yunnanensis,T. wallichiana,T. cuspidata, andT. media. Chemometric methods, including hierarchical cluster analysis, principal component analysis, orthogonal partial least squares-discriminate analysis, random forest iterative modeling, and fisher linear discriminant analysis, were utilized to differentiate and evaluate the sixTaxusspecies.Results:This proposed method exhibited good linearity (R2= 0.9999–0.9972) with a lower quantification limits of 0.94–3.05 ng/mL for all analytes. The intra- and inter-day precisions were within 6.83%. Six compounds, namely, 7-xylosyl-10-deacetyltaxol, ginkgetin, rutin, aromadendrin, 10-deacetyl baccatin III, and epigallocatechin, were identified through chemometrics for the first time. These compounds can be used as important chemical markers to distinguish the above sixTaxusspecies rapidly.Conclusion:This study established a method for determination of the leaves of sixTaxusspecies, and revealing the differences in the chemical components of these sixTaxusspecies.