Recently, 5-hydroxymethyl cytosine (5hmC) was identified in higher organisms as a novel epigenetic modification factor, and was found to be substantially enriched in the central nervous system relative to many other tissues and cell types. Additionally, epigenetic modifications are markedly involved in many neurological disorders. However, the precise role of 5hmC in the brain and neurological diseases remains elusive. To reveal its functional role, a general screen of its spatial and temporal distribution was proposed as being a reasonable starting investigation. Here, we found that 5hmC was widely distributed in the cerebral cortex, striatum, hippocampus, cerebellum, and the brain stem. At the cellular level, 5hmC was widely expressed in neurons and astrocytes even probably the majority of glial cells. Further, the content of 5hmC in different brain regions was inconsistent. Moreover, the pattern of 5hmC in the regions of the whole rat brain was highly susceptible to age-associated modifications. We also found similar phenomena in the striatum, which had not been previously studied. Also, unlike other brain regions, for example in the cerebellum and granulosa cells, 5hmC also appeared to display specific expression in these tissues. However, we didn’t obtain the expected result that 5hmC will be increased in 6-hydroxydopamine-induced models of Parkinson’s disease with regard the preliminary exploration of 5hmC in these models. Our results suggest that in rats and other mammals, 5hmC likely plays an important role in the brain and is associated with neural development and aging in different areas of the brain.