The crucial role of aerobic energy production in sustaining eukaryotic life positions mitochondrial processes as key determinants of an animal's ability to withstand unpredictable environments. The advent of new techniques facilitating the measurement of mitochondrial function offers an increasingly promising tool for conservation approaches. Herein, we synthesize the current knowledge on the links between mitochondrial bioenergetics, ecophysiology and local adaptation, expanding them to the wider conservation physiology field. We discuss recent findings linking cellular bioenergetics to whole-animal fitness, in the current context of climate change. We summarize topics, questions, methods, pitfalls and caveats to help provide a comprehensive roadmap for studying mitochondria from a conservation perspective. Our overall aim is to help guide conservation in natural populations, outlining the methods and techniques that could be most useful to assess mitochondrial function in the field.