SUMMARY In high-altitude vertebrates, adaptive changes in blood–O 2 affinity may be mediated by modifications of hemoglobin (Hb) structure that affect intrinsic O 2 affinity and/or responsiveness to allosteric effectors that modulate Hb–O 2 affinity. This mode of genotypic specialization is considered typical of mammalian species that are high-altitude natives. Here we investigated genetically based differences in Hb–O 2 affinity between highland and lowland populations of the deer mouse ( Peromyscus maniculatus ), a generalist species that has the broadest altitudinal distribution of any North American mammal. The results of a combined genetic and proteomic analysis revealed that deer mice harbor a high level of Hb isoform diversity that is attributable to allelic polymorphism at two tandemly duplicated α-globin genes and two tandemly duplicated β-globin genes. This high level of isoHb diversity translates into a correspondingly high level of interindividual variation in Hb functional properties. O 2 equilibrium experiments revealed that the Hbs of highland mice exhibit slightly higher intrinsic O 2 affinities and significantly lower Cl – sensitivities relative to the Hbs of lowland mice. The experiments also revealed distinct biochemical properties of deer mouse Hb related to the anion-dependent allosteric regulation of O 2 affinity. In conjunction with previous findings, our results demonstrate that modifications of Hb structure that alter allosteric anion sensitivity play an important role in the adaptive fine-tuning of blood–O 2 affinity.