Tuberous roots of Ophiopogon japonicus and Liriope spicata are collectively used as maidong medicine in China for the same clinical efficacy, nourish yin and generate fluids, moisten lung, and clear heart fire. Extensive cultivation of these species has led to the need for more robust quality assessments. Nevertheless, few studies have characterized the metabolomic profile specific to species and growing regions, with a particular focus on their active compounds. A comprehensive understanding of metabolomic profiles of maidong is essential to guide quality control efforts effectively. Therefore, an untargeted metabolomics profiling approach was conducted using ultra-high performance liquid chromatography coupled to a timsTOF Pro hybrid quadrupole-time-of-flight mass spectrometer. Compounds were annotated using an integrated approach based on feature-based molecular networking (FBMN) together with in silico and experimental spectra databases comparisons. The present study showcases a holistic overview of the metabolomic diversity and variation among maidong derived from different origins. Steroidal saponins and homoisoflavonoids were recognized as predominant chemical classes in L. spicata, and O. japonicus, respectively. Characteristic metabolites among maidong derived from four origins were highlighted. Annotations of 58 metabolites revealed significant inter-species discrimination, with 6 and 36 metabolites critical for regional differentiation in L. spicata and O. japonicus, respectively. This approach supports the advancement of quality control strategies for botanical medicines, particularly those derived from multiple origins, ensuring a more rigorous chemical marker selection for botanical medicines.