Changes in environmental temperature can pose considerable challenges to animals and shifts in thermal habitat have been shown to be a major force driving species’ adaptation. These adaptations have been the focus of major research efforts to determine the physiological or metabolic constraints related to temperature and to reveal the phenotypic characters that can or should adjust. Considering the current consensus on climate change, the focus of research will likely shift to questioning whether ectothermic organisms will be able to survive future modifications of their thermal niches. Organisms can adjust to temperature changes through physiological plasticity (e.g., acclimation), genetic adaptation, or via dispersal to more suitable thermal habitats. Thus, it is important to understand what genetic and phenotypic attributes—at the individual, population, and species levels—could improve survival success. These issues are particularly important for ectotherms, which are in thermal equilibrium with the surrounding environment. To start addressing these queries, we should consider what physiological or metabolic functions are responsible for the impact of temperature on organisms. Some recent developments indicate that mitochondria are key metabolic structures determining the thermal range that an organism can tolerate. The catalytic capacity of mitochondria is highly sensitive to thermal variation and therefore should partly dictate the temperature dependence of biological functions. Mitochondria contain a complex network of different enzymatic reaction pathways that interact synergistically. The precise regulation of both adenosine triphosphate (ATP) and reactive oxygen species (ROS) production depends on the integration of different enzymes and pathways. Here, we examine the temperature dependence of different parts of mitochondrial pathways and evaluate the evolutionary challenges that need to be overcome to ensure mitochondrial adaptations to new thermal environments.