Myoglobin (Mb) buffers intracellular O₂ and facilitates diffusion of O₂ through the cell. These functions of Mb will be most effective when intracellular PO₂ is near the partial pressure of oxygen at which Mb is half saturated (P₅₀) of the molecule. We test the hypothesis that Mb oxygen affinity has evolved such that it is conserved when adjusted for body temperature among closely related animals. We measure oxygen P₅₀s tonometrically and oxygen dissociation rate constants with stopped flow and generate amino acid sequence from cDNA of Mbs from fish with different body temperatures. P₅₀s for the endothermic bluefin tuna, skipjack tuna, and blue marlin at 20°C were 0.62 ± 0.02, 0.59 ± 0.01, 0.58 ± 0.04 mmHg, respectively, and were significantly lower than those for ectothermic bonito (1.03 ± 0.07 mmHg) and mackerel (1.39 ± 0.03 mmHg). Because the oxygen affinity of Mb decreases with increasing temperature, the above differences in oxygen affinity between endothermic and ectothermic fish are reduced when adjusted for the in vivo muscle temperature of the animal. Oxygen dissociation rate constants at 20°C for the endothermic species ranged from 34.1 to 49.3 s¹, whereas those for mackerel and bonito were 102 and 62 s¹, respectively. Correlated with the low oxygen affinity and fast dissociation kinetics of mackerel Mb is a substitution of alanine for proline that would likely result in a more flexible mackerel protein.