Habitats vary in temperature both spatially and temporally. Variation in thermal habitat introduces challenges to organisms and may reduce fitness unless organisms can physiologically adjust to such changes. Theory predicts that thermal variability should influence the capacity for acclimation such that increased variation should favor a reduction in the thermal sensitivity of physiological traits. In this study, we investigated acclimation to constant and variable conditions in populations of the salamander Desmognathus brimleyorum from the Ouachita Mountains of Arkansas, USA. We exposed salamanders to constant and variable temperature regimes for 8 weeks in the laboratory. We then tested salamanders for acclimation of thermal tolerance, and the thermal sensitivities of swimming performance and standard metabolic rate. Our results indicate limited capacity for thermal acclimation to constant and variable conditions in D. brimleyorum. Instead, variation in physiological traits is dominated by differences among populations. Population differences do not appear to be correlated with observed variation in the thermal conditions of the streams, but are likely a consequence of structural and ecological differences. Due to the mixed support for theoretical predictions for acclimation to alternative environments, further consideration should be given to revising and expanding current theoretical models.