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Introduction: Epimedium L., the largest herbaceous genus of Berberidaceae, is one of the most taxonomically difficult representatives. The classification and phylogenetic relationships within Epimedium are controversial and unresolved., Objectives: For the first time, we systematically studied the phylogeny and evolution of Epimedium based on plastid genome (plastome) data for better understanding this enigmatic genus., Methods: We explored the molecular phylogeny, assessed the infrageneric classification, estimated the divergence times, and inferred the ancestral states for flower traits of Epimedium based on 45 plastomes from 32 species., Results: The Epimedium plastome length ranged from 156,635 bp to 159,956 bp. Four types of plastome organization with different inverted repeat boundary changes were identified. Phylogenetic analysis revealed a strong support for the sister relationship of sect. Macroceras and sect. Diphyllon but did not provide a distinct route for petal evolution in sect. Diphyllon . Disharmony between phylogenetic relationships and traditional classification of sect. Diphyllon was observed. Results from divergence time analysis showed that Epimedium diverged in the early Pleistocene (~2.11 Ma, 95% HPD = 1.88-2.35 Ma). Ancestral character state reconstructions indicated transitions from long spur (large-flowered group) to other petal types (small-flowered group) in Epimedium ., Conclusion: These findings provide new insights into the relationships among Epimedium species and pave the way for better elucidation of the classification and evolution of this genus., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors. Published by Elsevier B.V. on behalf of Cairo University.)