BackgroundSemen Aesculi, a traditional Chinese herbal medicine, has a long history of use for treating chest and abdominal pain with distension. In addition, the horse chestnut (Aesculus hippocastanum L.) is another species of Aesculus in Europe and has notable clinical significance in alleviating chronic venous insufficiency, hemorrhoids, and postoperative edema. Thus, highlighting the comparative study of Semen Aesculi and horse chestnut may broaden clinical applications.ObjectivesTo conduct a comprehensive comparative analysis on the chemical profiling of these two varieties and determine whether they have equivalent clinical efficacy by integrating plant metabolomics and multivariate statistical methods.MethodsInitially, a comprehensive characterisation was performed using ultra‐performance liquid chromatography quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐QTOF‐MS/MS) platform, and in total 44 active ingredients were identified. Then, untargeted metabolomics combined with principal component analysis (PCA) and partial least squares‐discriminant analysis (PLS‐DA) was applied for the discrimination of a German species and three official Chinese species. Next, 24 marker compounds responsible for the discrimination of different species were screened out and used to predict the species of unknown samples by genetic algorithm‐optimised support vector machine (GA‐SVM) with a high prediction accuracy. Finally, a heatmap visualisation was employed for clarifying the distribution of the identified active ingredients.ResultsThe three species of Chinese Semen Aesculi showed distinct separation from each other, while European horse chestnut and Aesculus chinensis Bunge were similar in chemical composition.ConclusionsThis work provided experimental evidence for further expanding the clinical application of Chinese Semen Aesculi and promoted the species identification and quality control of Semen Aesculi.