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Mitochondria provide the vast majority of cellular energy available to eukaryotes. Therefore, adjustments in mitochondrial function through genetic changes in mitochondrial or nuclear-encoded genes might underlie environmental adaptation. Environmentally induced plasticity in mitochondrial function is also common, especially in response to thermal acclimation in aquatic systems. Here, we examined mitochondrial function in mayfly larvae ( Baetis and Drunella spp.) from high and low elevation mountain streams during thermal acclimation to ecologically relevant temperatures. A multi-substrate titration protocol was used to evaluate different respiratory states in isolated mitochondria, along with cytochrome oxidase and citrate synthase activities. In general, maximal mitochondrial respiratory capacity and oxidative phosphorylation coupling efficiency decreased during acclimation to higher temperatures, suggesting montane insects may be especially vulnerable to rapid climate change. Consistent with predictions of the climate variability hypothesis, mitochondria from Baetis collected at a low elevation site with highly variable daily and seasonal temperatures exhibited greater thermal tolerance than Baetis from a high elevation site with comparatively stable temperatures. However, mitochondrial phenotypes were more resilient than whole-organism phenotypes in the face of thermal stress. These results highlight the complex relationships between mitochondrial and organismal genotypes, phenotypes and environmental adaptation. This article is part of the theme issue 'Linking the mitochondrial genotype to phenotype: a complex endeavour'.