Both substrate availability and animal selectivity can contribute to patterns of animal substrate use. In the West Indies, where Anolis lizard ecomorphs are specialized for particular microhabitats, the relative influences of availability and selectivity on anole substrate use can change depending on the species and its location. Whether substrate availability and selectivity both contribute to microhabitat specialization in anoles outside the West Indies is largely unexplored. Two species of Central American semiaquatic anoles appear adapted for locomotion on different substrates—Anolis oxylophus on wood and leaves and Anolis aquaticus on rocks. I evaluated the contributions of substrate availability and selectivity to microhabitat specialization in these two species by comparing their substrate use to substrate availabilities in their stream macrohabitats. Both species selectively avoided the ground and perched instead on elevated substrates, yet the available elevated substrates differed between streams. Specifically, wood was most abundant in streams occupied by A. oxylophus, and rocks were most abundant in streams occupied by A. aquaticus. As is true with several West Indian anole ecomorphs, both substrate availability and selectivity contribute to microhabitat specialization in A. oxylophus and A. aquaticus.