Phenotypic plasticity can contribute to the process of adaptive radiation by facilitating population persistence in novel environments. West Indian Anolis lizards provide a classic example of an adaptive radiation, in which divergence has occurred along two primary ecological axes: structural microhabitat and climate. Adaptive plasticity in limb morphology is hypothesized to have facilitated divergence along the structural niche axis in Anolis, but very little work has explored plasticity in physiological traits. Here, we experimentally ask whether Puerto Rican Anolis cristatellus from mesic and xeric habitats differ in desiccation rates, and whether these lizards exhibit an acclimation response to changes in relative humidity. We first present microclimatic data collected at lizard perch sites that demonstrate that abiotic conditions experienced by lizards differ between mesic and xeric habitat types. In Experiment 1, we measured desiccation rates of lizards from both habitats maintained under identical laboratory conditions. This experiment demonstrated that desiccation rates differ between populations; xeric lizards lose water more slowly than mesic lizards. In Experiment 2, lizards from each habitat were either maintained under the conditions of Experiment 1, or under extremely low relative humidity. Desiccation rates did not differ between lizards from the same habitat maintained under different treatments and xeric lizards maintained lower desiccation rates than mesic lizards within each treatment. Our results demonstrate that A. cristatellus does not exhibit an acclimation response to abrupt changes of hydric conditions, and suggest that tropical Anolis lizards might be unable to exhibit physiological plasticity in desiccation rates in response to varying climatic conditions.