Myoglobin (Mb) is an oxygen binding protein found in vertebrate skeletal muscle, where it facilitates intracellular transport and storage of oxygen. This protein has evolved to suit unique physiological needs in the muscle of diving vertebrates that express Mb at much greater concentrations than their terrestrial counterparts. In this study, we characterized Mb oxygen affinity (P 50 ) from 25 species of aquatic and terrestrial birds and mammals. Among diving species, we tested for correlations between Mb P 50 and routine dive duration. Across all species examined, Mb P 50 ranged from 2.40 to 4.85 mmHg. The mean P 50 of Mb from terrestrial ungulates was 3.72±0.15 mmHg (range 3.70-3.74 mmHg). The P 50 of cetaceans was similar to terrestrial ungulates ranging from 3.54 to 3.82 mmHg, with the exception of the melonheaded whale, which had a significantly higher P 50 of 4.85 mmHg. Among pinnipeds, the P 50 ranged from 3.23 to 3.81 mmHg and showed a trend for higher oxygen affinity in species with longer dive durations. Among diving birds, the P 50 ranged from 2.40 to 3.36 mmHg and also showed a trend of higher affinities in species with longer dive durations. In pinnipeds and birds, low Mb P 50 was associated with species whose muscles are metabolically active under hypoxic conditions associated with aerobic dives. Given the broad range of potential globin oxygen affinities, Mb P 50 from diverse vertebrate species appears constrained within a relatively narrow range. High Mb oxygen affinity within this range may be adaptive for some vertebrates that make prolonged dives.