Ecology and adaptive differentiation of Epimedium are central to understanding both its taxonomic complexity and medicinal value. In this study, we integrate transcriptomic and plastid data from four natural populations of E. brevicornu (HZ, QLH, TS, WD) to reconstruct their phylogenetic relationships, estimate divergence times, and identify candidate genes associated with local adaptation. Nuclear gene-based phylogenies provide higher resolution and greater topological consistency than plastid data, underscoring the utility of nuclear data in lineages affected by hybridization and incomplete lineage sorting. Molecular dating indicated that major intraspecific divergence occurred during the mid-Quaternary (0.61–0.45 Ma), coinciding with climatic oscillations and montane isolation. Population structure showed strong correlations with temperature and precipitation gradients, suggesting environmentally driven selection. Signatures of positive selection and accelerated evolutionary rates revealed population-specific enrichment of genes involved in stress response, protein modification, signaling, and carbohydrate metabolism—key pathways linked to high-elevation adaptation. Protein–protein interaction networks further indicated a two-tier adaptation mechanism: ancestral network rewiring combined with population co-evolution of interacting genes. Together, these findings advance our understanding of alpine plant adaptation and provide candidate genes for further functional and breeding studies in Epimedium.