Clarifying the role of key genes in the early stage of speciation is critical to understand the origin and evolution of species. Aquilegia viridiflora, in the early stage of speciation, has a wide distribution range, and shows obvious phenotypic variation among its different populations. Here, we analyzed the adaptive evolution mechanism of phenotypic differences in the early stages of speciation using the phenotypes, genomes, and transcriptomes of different populations of A. viridiflora. Our results indicated that A. viridiflora originated in northwestern China, and the environmental changes caused by the uplift of the northeastern Qinghai–Tibet Plateau in the late Miocene may have caused its differentiation. Within its distribution range, flower size was significantly negatively correlated with the inflorescence number and leaf area. Aqcoe5G459400 expression was reduced in environments with large temperature differences between day and night, possibly resulting in longer spurs and promoting the formation of reproductive isolation. Additionally, the cytochrome P450 (CYP 450) superfamily may have driven species differentiation in the early stage of speciation. Our study reveals the genetic basis of the adaptive evolution of the phenotype in the early stage of speciation and provides new evidence of the rapid evolution of angiosperms.