AbstractThe study of the hypothalamus and its topological changes provides valuable insights into underlying physiological and pathological processes. Owing to technological limitations, however, in vivo atlases detailing hypothalamic anatomy are currently lacking in the literature. In this work we aim to overcome this shortcoming by generating a high-resolution in vivo anatomical atlas of the human hypothalamic region. A minimum deformation averaging (MDA) pipeline was employed to produce a normalized, high-resolution template from multimodal magnetic resonance imaging (MRI) datasets. This template was used to delineate hypothalamic (n = 13) and extrahypothalamic (n = 12) gray and white matter structures. The reliability of the atlas was evaluated as a measure for voxel-wise volume overlap among raters. Clinical application was demonstrated by superimposing the atlas into datasets of patients diagnosed with a hypothalamic lesion (n = 1) or undergoing hypothalamic (n = 1) and forniceal (n = 1) deep brain stimulation (DBS). The present template serves as a substrate for segmentation of brain structures, specifically those featuring low contrast. Conversely, the segmented hypothalamic atlas may inform DBS programming procedures and may be employed in volumetric studies.