Most histones are assembled into nucleosomes during replication to package genomic DNA. However, several variant histones are deposited independently of replication at particular regions of chromosomes. Such histone variants include cenH3, which forms the nucleosomal foundation for the centromere, and H3.3, which replaces histones that are lost during dynamic processes that disrupt nucleosomes. Furthermore, various H2A variants participate in DNA repair, gene regulation and other processes that are, as yet, not fully understood. Here, we review recent studies that have implicated histone variants in maintaining pluripotency and as causal factors in cancer and other diseases.