The oxygen binding characteristics of recombinant hemoglobin isoforms (HbA [α2β2] and HbA2 [α2δ2]) from the woolly rhinoceros (Coelodonta antiquitatis) are compared with Sumatran rhinoceros (Dicerorhinus sumatrensis), black rhinoceros (Diceros bicornis), and horse (Equus caballus). Oxygen equilibrium curves were determined in the presence and absence of allosteric effectors at 25 and 37°C. Horse HbA low temperature sensitivity was primarily driven by increased DPG binding at the lower test temperature. The major HBA woolly rhino isoform possessed nearly identical inherent oxygen affinities, allosteric effector sensitivities, and thermal sensitivities to those of the extant rhinoceros species. The woolly rhino evolved a rare residue replacement at (δ104Arg→Ser) which abolishes DPG binding to woolly rhino HbA2 while also dramatically reducing the effects of both pH and Cl- on Hb–O2 affinity. Thus, the extinct woolly rhino did not evolve cold-adapted hemoglobin which argues against strong regional heterothermy in this species.