5‐Methylcytosine (5mC) in genomic DNA has important epigenetic functions in embryonic development and tumor biology. 5‐Hydroxymethylcytosine (5hmC) is generated from 5mC by the action of the TET (Ten‐Eleven‐Translocation) enzymes and may be an intermediate to further oxidation and finally demethylation of 5mC. We have used immunohistochemistry (IHC) and isotope‐based liquid chromatography mass spectrometry (LC‐MS) to investigate the presence and distribution of 5hmC in human brain and brain tumors. In the normal adult brain, IHC identified 61.5% 5hmC positive cells in the cortex and 32.4% 5hmC in white matter (WM) areas. In tumors, positive staining of cells ranged from 1.1% in glioblastomas (GBMs) (WHO Grade IV) to 8.9% in Grade I gliomas (pilocytic astrocytomas). In the normal adult human brain, LC‐MS also showed highest values in cortical areas (1.17% 5hmC/dG deoxyguanosine), in the cerebral WM we measured around 0.70% 5hmC/dG. 5hmC levels were related to tumor differentiation, ranging from lowest values of 0.078% 5hmC/dG in GBMs (WHO Grade IV) to 0.24% 5hmC/dG in WHO Grade II diffuse astrocytomas. 5hmC measurements were unrelated to 5mC values. We find that the number of 5hmC positive cells and the amount of 5hmC/dG in the genome that has been proposed to be related to pluripotency and lineage commitment in embryonic stem cells is also associated with brain tumor differentiation and anaplasia.