AimDescription of the driving forces for genetic divergence is important for understanding spatial pattern of biodiversity and development of conservation plans. Paleo‐climate, geographical barriers and habitat heterogeneity are considered to be the main influential factors; however, an integrative study is still lacking to reveal their interactions.LocationEastern North America.MethodsHere, we generated MIG‐seq SNPs of 403 Sanguinaria canadensis samples and seven plastome data. The methods of phylogeography and landscape genomics were applied to infer their genetic divergence, demography, species distribution modelling, ecological differentiation and local adaptation.ResultsWe identified three distinct genetic lineages corresponding to geographical distributions isolated by the Appalachian Mountains and the Mississippi River. Ecological niche modelling and population demographic inference demonstrated that the response of S. canadensis to Pleistocene climate changes was consistent with the pattern of southward contraction during the ice age and northward recolonization during the inter−/postglacial period. Isolation in multiple southern refugia was a key factor resulting in the genetic divergence, whereas secondary contact triggered by repeated range shifts allowed gene flow among different lineages. A greater effect of isolation‐by‐environment than isolation‐by‐distance was founded, which suggested heterogeneous environment was also a promotor of genetic differentiation. Candidate adaptive loci related to transposable elements were identified under the influence of divergent environmental selection. Ecological niche divergence also reflected adaptation to different environments.Main ConclusionsThis study revealed that genetic divergence has occurred within S. canadensis populations under the combined effect of paleo‐climate, geographical barriers and habitat heterogeneity, and emphasized the necessity of establishing different conservation units in future biological conservation and management work.