Molecular evolutionary analyses revealed that histone H2A.Bbd is a highly variable quickly evolving mammalian replacement histone variant, in striking contrast to all other histones. At the nucleotide level, this variability appears to be the result of a larger amount of nonsynonymous variation, which affects to a lesser extent, the structural domain of the protein comprising the histone fold. The resulting amino acid sequence diversity can be predicted to affect the internucleosomal and intranucleosomal histone interactions. Our phylogenetic analysis has allowed us to identify several of the residues involved. The biophysical characterization of nucleosomes reconstituted with recombinant mouse H2A.Bbd and their comparison to similar data obtained with human H2A.Bbd clearly support this notion. Despite the high interspecific amino acid sequence variability, all of the H2A.Bbd variants exert similar structural effects at the nucleosome level, which result in an unfolded highly unstable nucleoprotein complex. Such structure resembles that previously described for the highly dynamically acetylated nucleosomes associated with transcriptionally active regions of the genome. Nevertheless, the structure of nucleosome core particles reconstituted from H2A.Bbd is not affected by the presence of a hyperacetylated histone complement. This suggests that replacement by H2A.Bbd provides an alternative mechanism to unfold chromatin structure, possibly in euchromatic regions, in a way that is not dependent on acetylation.—Eirín-López, J. M., Ishibashi, T., and Ausió, J. H2A.Bbd: a quickly evolving hypervariable mammalian histone that destabilizes nucleosomes in an acetylation-independent way.