The eukaryotic genome exists in vivo at an equimolar ratio with histones, thus forming a polymer composed of DNA and histone proteins. Each nucleosomal unit in this polymer provides versatile capabilities and dynamic range. Substitutions of the individual components of the histone core with structurally distinct histone variants and covalent modifications alter the local fabric of the chromatin fiber, resulting in epigenetic changes that can be regulated by the cell. In this review, we highlight recent advances in the study of histone variant structure, assembly, and inheritance, their influence on nucleosome positioning, and their cumulative effect upon gene expression, DNA repair and the progression of disease. We also highlight fundamental questions that remain unanswered regarding the behavior of histone variants and their influence on cellular function in the normal and diseased states.