Enhancer of Zeste Homolog 2 (EZH2) plays an essential epigenetic role in Diffuse Large B Cell Lymphoma (DLBCL) development. Recurrent somatic heterozygous gain-of-function mutations of EZH2 have been identified in DLBCL, most notably affecting tyrosine 641 (Y641), inducing hyper-trimethylation of H3K27 (H3K27me3). Novel EZH2 inhibitors are being tested in phase 1 and 2 clinical trials but no study has examined which patients would most benefit from this treatment. We evaluated the immunohistochemical (IHC) methylation profiles of 82 patients with DLBCL, as well as the mutational profiles of 32 patients with DLBCL using NGS analysis of a panel of 34 genes involved in lymphomagenesis. A novel IHC score based on H3K27me2 and H3K27me3 expression was developed, capable of distinguishing patients with wild-type (WT) EZH2 and patients with EZH2 Y641 mutations (p = 10-5). NGS analysis revealed a subclonal EZH2 mutation pattern in EZH2 mutant patients with WT-like IHC methylation profiles, while associated mutations capable of upregulating EZH2 were detected in WT EZH2 patients with mutant-like IHC methylation profiles. IHC and mutational profiles highlight in vivo hyper-H3K27me3 and hypo-H3K27me2 status, pinpoint associated activating mutations and determine EZH2 mutation clonality, maximizing EZH2 inhibitor potential by identifying patients most likely to benefit from treatment.