AbstractModification of nuclear and cytoplasmic proteins by the addition or removal of O‐GlcNAc dynamically impacts multiple biological processes. Here, we present the development of a chemoenzymatic histology method for the detection of O‐GlcNAc in tissue specimens. We applied this method to screen murine organs, uncovering specific O‐GlcNAc distribution patterns in different tissue structures. We then utilized our histology method for O‐GlcNAc detection in human brain specimens from healthy donors and donors with Alzheimer's disease and found higher levels of O‐GlcNAc in specimens from healthy donors. We also performed an analysis using a multiple cancer tissue array, uncovering different O‐GlcNAc levels between healthy and cancerous tissues, as well as different O‐GlcNAc cellular distributions within certain tissue specimens. This chemoenzymatic histology method therefore holds great potential for revealing the biology of O‐GlcNAc in physiopathological processes.