Determining whether organisms have preferences for certain aspects of the habitat is important for understanding the process of habitat selection. Anolis lizards (anoles) have evolved to occupy distinct parts of the arboreal environment. Depending on the population or species, anoles perch on different aspects of the structural habitat varying in diameter, height, and type of substrate. We used laboratory and mesocosm trials to assess whether 2 species of Anolis lizards have preferences for the diameter and type of substrate they use for perching. Results reveal A. cristatellus and A. sagrei both prefer larger diameter perches. Lizards from natural forests and urban populations showed no difference in perch-diameter preference. In contrast, A. sagrei from an urban site overwhelmingly preferred to perch on wood substrates over polyvinyl chloride and metal ones. The larger diameter and wood substrates preferred by anoles in our trials should confer increased locomotor performance for these species based on previous studies. Rapid environmental change caused by humans can decouple the relationship between habitat preferences and fitness, so it is essential to test for changing preferences in organisms that experience urbanization and other forms of global change.