Secondary metabolites are usually the bioactive components of medicinal plants. The difference in the secondary metabolisms of closely related plant species and their hybrids has rarely been addressed. In this study, we conducted a holistic secondary metabolomics analysis of three medicinal Glycyrrhiza species (G. uralensis, G. glabra, and G. inflata), which are used as the popular herbal medicine licorice. The Glycyrrhiza species (genotype) for 95 batches of samples were identified by DNA barcodes of the internal transcribed spacer and trnV-ndhC regions, and the chemotypes were revealed by LC/UV- or LC/MS/MS-based quantitative analysis of 151 bioactive secondary metabolites, including 17 flavonoid glycosides, 24 saponins, and 110 free phenolic compounds. These compounds represented key products in the biosynthetic pathways of licorice. For the 76 homozygous samples, the three Glycyrrhiza species showed significant biosynthetic preferences, especially in coumarins, chalcones, isoflavanes, and flavonols. In total, 27 species-specific chemical markers were discovered. The 19 hybrid samples indicated that hybridization could remarkably alter the chemical composition and that the male parent contributed more to the offspring than the female parent did. This is hitherto the largest-scale targeted secondary metabolomics study of medicinal plants and the first report on uniparental inheritance in plant secondary metabolism. The results are valuable for biosynthesis, inheritance, and quality control studies of licorice and other medicinal plants.