Maintaining energy balance over a wide range of temperatures is critical for an active pelagic fish species such as the mahi-mahi (Coryphaena hippurus), which can experience rapid changes in temperature during vertical migrations. Due to the profound effect of temperature on mitochondrial function, this study was designed to investigate the effects of temperature on mitochondrial respiration in permeabilized heart and red skeletal muscle (RM) fibres isolated from mahi-mahi. As RM is thought to be more anatomically isolated from rapid ambient temperature changes compared to the myocardium, it was hypothesized that heart mitochondria would be more tolerant of temperature changes through a greater ability to match respiratory capacity to an increase in temperature and to maintain coupling, when compared to RM mitochondria. Results show that heart fibres were more temperature sensitive and increased respiration rate with temperature increases to a greater degree than RM. Respiratory coupling ratios at the three assay temperatures (20, 26, and 30 °C), revealed that heart mitochondria were less coupled at a lower temperature (26 °C) compared to RM mitochondria (30 °C). In response to an in vitro acute temperature challenge, both tissues showed irreversible effects, where both heart and RM increased uncoupling whether the assay temperature was acutely changed from 20 to 30 °C or 30 to 20 °C. The findings from this study indicate that mahi-mahi heart mitochondria were more temperature sensitive compared to those from RM.