Abstract The evolution of metabolic rate-temperature (MR-T) reaction norms is of fundamental importance to physiological ecology. Metabolic cold adaptation (MCA) predicts that populations or species from cooler environments will have either a higher metabolic rate at a common temperature or steeper MR-T relationships, indicating greater sensitivity of respiratory metabolism to temperature. Support for MCA has been found in some insect species by comparing species or populations differing in latitude. However, the generality of these findings are contentious, with most studies either unable to account for phenotypic plasticity or the evolutionary relatedness of species or populations. Hence, the importance of MCA is vigorously debated from both evolutionary and ecological perspectives. Furthermore, few species, particularly from tropical environments, have been shown to differ in MR-T sensitivity along altitudinal temperature gradients. Here, using four populations of tsetse flies (Glossina pallidipes, Diptera: Glossinidae) from thermally distinct geographic regions, we test the hypothesis that there is evolved variation in MR-T relationships to cold climates. We found that a high-altitude equatorial population from a cool habitat has a steeper MR-T reaction norm. By contrast, other populations from warmer environments in East Africa do not differ with respect to their MR-T reaction norms. Squared-change parsimony analyses, based on the combined mitochondrial 16S rDNA ribosomal subunit and cytochrome c oxidase subunit I (COI), support the hypothesis of adaptive differentiation of MR-T reaction norms in the cool-climate population. Seasonal adjustments or laboratory-temperature-induced phenotypic plasticity changed the intercept of the reaction norm rather than the slope, and thus the observed intraspecific variation in slopes of MR-T reaction norms could not be accounted for by phenotypic plasticity. These results therefore suggest evolutionary adaptation of MR-T reaction norms to cool climates (<22 degrees C) in tsetse and provide novel support for MCA within an insect species.