The ability of ectothermic animals to live in different thermal environments is closely associated with their capacity to maintain physiological homeostasis across diurnal and seasonal temperature fluctuations. For chill-susceptible insects, such as Drosophila, cold tolerance is tightly linked to ion and water homeostasis obtained through a regulated balance of active and passive transport. Active transport at low temperature requires a constant delivery of ATP and we therefore hypothesize that cold-adapted Drosophila are characterized by superior mitochondrial capacity at low temperature relative to cold-sensitive species. To address this, we investigated how experimental temperatures from 1 to 19°C affected mitochondrial substrate oxidation in flight muscle of seven Drosophila species and compared it with a measure of species cold tolerance (CTmin, the temperature inducing cold coma). Mitochondrial oxygen consumption rates measured using a substrate–uncoupler–inhibitor titration (SUIT) protocol showed that cooling generally reduced oxygen consumption of the electron transport system across species, as was expected given thermodynamic effects. Complex I respiration is the primary consumer of oxygen at non-stressful temperatures, but low temperature decreases complex I respiration to a much greater extent in cold-sensitive species than in cold-adapted species. Accordingly, cold-induced reduction of complex I respiration correlates strongly with CTmin. The relative contribution of other substrates (proline, succinate and glycerol 3-phosphate) increased as temperature decreased, particularly in the cold-sensitive species. At present, it is unclear whether the oxidation of alternative substrates can be used to offset the effects of the temperature-sensitive complex I, and the potential functional consequences of such a substrate switch are discussed.