The Channel Islands off the coast of southern California host >50 species of terrestrial vertebrates with varying degrees of phenotypic differentiation. However, most organisms that breed on the Channel Islands remain unstudied with respect to genetic differentiation from mainland populations. By comparing patterns of genetic variation between the Channel Islands and the mainland, we aimed to further our understanding of the role that the Channel Islands have played in diversification of the North American biota. We evaluated long-standing, untested hypotheses regarding colonization patterns and evolutionary relationships among western populations of the Horned Lark (Eremophila alpestris), including the endemic Channel Island subspecies E. a. insularis. We also examined how many times Horned Larks have colonized the Channel Islands, whether the species exhibits asymmetrical patterns of gene flow between mainland and island populations, and whether E. a. strigata of the Pacific Northwest is closely related to the phenotypically similar, but geographically separated, island subspecies. We found that E. a. insularis is polyphyletic, which suggests either multiple colonization events from the mainland or incomplete lineage sorting of a large ancestral population. We also inferred higher rates of migration from the Channel Islands to the mainland, with E. a. strigata being closely related to individuals from the Channel Islands and coastal southern California. Moreover, ecological niche models for E. a. strigata identified suitable abiotic conditions in southern California and the Pacific Northwest during the Last Glacial Maximum, which suggests that E. a. strigata experienced a postglacial range shift in addition to a population bottleneck. Our results provide novel insight regarding the origins of the Channel Island avifauna and the evolutionary history of the Horned Lark in the western United States. Moreover, our findings suggest that Channel Island birds may be weakly differentiated from mainland populations despite phenotypic differences between recognized subspecies.