Summary1. Within-individual plasticity (acclimation) counteracts potentially negative physiological effects resulting from environmental changes and thereby maintains fitness across a broad range of environments. The capacity for the acclimation of individuals may therefore determine the persistence of populations in variable environments. 2. We determined phylogenetic relationships by Amplified Fragment Length Polymorphism (AFLP) analysis of six populations of mosquitofish (Gambusia holbrooki) from coastal and mountain environments and compared their capacity for thermal acclimation to test the hypotheses that acclimation capacity is greater in more seasonal environments with less diurnal variability, that acclimation is genetically constrained and that demographic processes determine acclimation capacity. 3. We show that populations are divided into distinct genetic lineages and that populations within lineages have distinct genetic identities. There were significant differences in the capacity for acclimation between traits (swimming performance, citrate synthase and lactate dehydrogenase activities), between lineages and between populations within lineages. 4. We rejected the hypothesis that climatic conditions (coastal vs. mountain) determined the capacity for acclimation, but accepted the hypotheses that demographic processes and genetic constraint influenced thermal acclimation. 5. The importance of our data lies in proof of concept that there can be substantial variation in thermal plasticity between populations within species. Similar responses are likely to be found in other species that comprise structured populations. Many predictions of the impact of climate change on biodiversity assume a species-specific response to changing environments. Based on our results, we argue that this resolution can be too coarse and that analysis of the impacts of climate change and other environmental variability should be resolved to a population level.