<p>Phenotypic diversity in plants reflects genetic variation among different germplasms. <i>Astragalus mongholicus</i> displays two distinct stem color phenotypes: green and purplish-red. However, the regulatory mechanisms underlying this color variation and its impact on pharmacologically active compound accumulation remain poorly understood. In this study, we integrated non‑targeted metabolomics of roots and stems with transcriptomics of stems and performed HPLC analysis to investigate the molecular basis of stem color differentiation. Our results indicated that the flavonoid biosynthesis pathway significantly enriched the differentially accumulated metabolites (DAMs) in both stems and roots of the two phenotypes. Integrated transcriptomic and metabolomic analysis of stems further revealed significant upregulation of key anthocyanin biosynthetic genes (<i>ANS</i>, <i>DFR1, CHS2</i>, <i>CHS3</i>, and <i>CHS5)</i> in purplish‑red stems (PS). This upregulation was associated with enhanced accumulation of specific anthocyanins, including delphinidin 3-lathyroside-5-glucoside, delphinidin 3-sambubioside, and malvidin 3-O-galactoside. Consistently, HPLC analysis confirmed that the contents of major bioactive constituents were significantly lower in purplish‑red stems and their corresponding roots compared with the green stems (GS) phenotype. These constituents included calycosin-7-O-<i>β</i>-D-glucoside, calycosin, and formononetin. In conclusion, the PS phenotype showed much lower levels of bioactive constituent compounds during peak vegetative growth than the GS phenotype. These results suggest that the coordinated upregulation of the PS anthocyanin pathway may constitute a core mechanism coordinating stem color variation and accumulation of medicinal components in <i>A. mongholicus</i>. These findings provide molecular insights into the regulation of stem pigmentation and offer valuable guidance for germplasm selection and medicinal quality evaluation.</p>