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 (P50) from 25 species of aquatic and terrestrial birds and mammals. Among diving species we tested for correlations between Mb P50 and routine dive duration. Across all species examined, Mb P50 ranged from 2.40–4.85 mmHg. The mean P50 of Mb from terrestrial ungulates was 3.72±0.15 mmHg (range 3.70–3.74 mmHg). The P50 of cetaceans was similar to terrestrial ungulates ranging from 3.54–3.82 mmHg with the exception of the melon-headed whale that had a significantly higher P50 of 4.85 mmHg. Among pinnipeds, the P50 ranged from 3.23–3.81 mmHg and showed a trend for higher oxygen affinity in species with longer dive durations. Among diving birds, the P50 ranged from 2.40–3.36 mmHg and also showed a trend of higher affinities in species with longer dive durations. In pinnipeds and birds, low Mb P50 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 P50 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.