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Abstract The genomic characteristics of adaptively radiated groups could contribute to their high species number and ecological disparity, by increasing their evolutionary potential. Here, we explored the genomic variation of Anolis lizards, focusing on three species with distinct phenotypes: A. auratus, one of the species with the longest tail; A. frenatus, one of the largest species; and A. carolinensis, one of the species that inhabits the coldest environments. We assembled and annotated two new chromosome-level reference genomes for A. auratus and A. frenatus, and compared them with the available genomes of A. carolinensis and A. sagrei. We evaluated the presence of structural rearrangements, quantified the density of repeat elements, and identified potential signatures of positive selection in coding and regulatory regions. We detected substantial rearrangements in scaffolds 1, 2 and 3 of A. frenatus different from the other species, in which the rearrangement breakpoints corresponded to hotspots of developmental genes. Further, we detected an accumulation of repeats around key developmental genes in anoles and phrynosomatid outgroups. Finally, coding sequences and regulatory regions of genes relevant to development and physiology showed variation that could be associated with the unique phenotypes of the analyzed species. Our results show examples of the hierarchical genomic variation within anoles, that could provide the substrate that promoted phenotypic disparity and contributed to their adaptive radiation.