Antarctic icefishes are the only vertebrates that do not have hemoglobin and erythrocytes in their blood. These startling phenotypes are associated in several icefish species with deletions of juvenile and adult globin loci, which in red-blooded teleosts are typically composed of tightly linked pairs of alpha- and beta-globin genes. It is unknown if the loss of hemoglobin expression in icefishes was the direct result of such deletions or if other mutational events compromised globin chain synthesis prior to globin gene loss. In this study, we show that 15 of the 16 icefish species have lost the adult beta-globin gene but retain a truncated alpha-globin pseudogene. Surprisingly, a phylogenetically derived icefish species, Neopagetopsis ionah, possesses a complete, but nonfunctional, adult alphabeta-globin complex. This cluster contains 2 distinct beta-globin pseudogenes whose phylogenetic origins span the entire Antarctic notothenioid radiation, consistent with an origin via introgression. Maximum likelihood ancestral state reconstruction supports a scenario of icefish globin gene evolution that involves a single loss of the transcriptionally active adult alphabeta-globin cluster prior to the diversification of the extant species in the clade. Through lineage sorting of ancestral polymorphism, 2 types of alleles became fixed in the clade: 1) the alpha-globin pseudogene of the majority of species and 2) the inactive alphabeta-globin complex of N. ionah. We conclude that the globin pseudogene complex of N. ionah is a "genomic fossil" that reveals key intermediate steps on the pathway to loss of hemoglobin expression by all icefish species.