Histone lysine (K) methylation has been shown to play a fundamental role in modulating chromatin architecture and regulation of gene expression. Here we report on the identification of histone H3K56, located at the pivotal, nucleosome DNA entry/exit point, as a novel methylation site that is evolutionary conserved. We identify trimethylation of H3K56 (H3K56me3) as a modification that is present during all cell cycle phases, with the exception of S-phase, where it is underrepresented on chromatin. H3K56me3 is a novel heterochromatin mark, since it is enriched at pericentromeres but not telomeres and is thereby similar, but not identical, to the localization of H3K9me3 and H4K20me3. Possibly due to H3 sequence similarities, Suv39h enzymes, responsible for trimethylation of H3K9, also affect methylation of H3K56. Similarly, we demonstrate that trimethylation of H3K56 is removed by members of the JMJD2 family of demethylases that also target H3K9me3. Furthermore, we identify and characterize mouse mJmjd2E and its human homolog hKDM4L as novel, functionally active enzymes that catalyze the removal of two methyl groups from trimethylated H3K9 and K56. H3K56me3 is also found in C. elegans, where it co-localizes with H3K9me3 in most, but not all, tissues. Taken together, our findings raise interesting questions regarding how methylation of H3K9 and H3K56 is regulated in different organisms and their functional roles in heterochromatin formation and/or maintenance.