OBJECTIVE The primary objective of this anatomical study was to apply innovative imaging techniques to increase understanding of the microanatomical structures of the brainstem related to safe entry zones. The authors hypothesized that such a high-detail overview would enhance neurosurgeons’ abilities to approach and define anatomical safe entry zones for use with microsurgical resection techniques for intrinsic brainstem lesions. METHODS The brainstems of 13 cadavers were studied with polarized light imaging (PLI) and 11.7-T MRI. The brainstem was divided into 3 compartments—mesencephalon, pons, and medulla—for evaluation with MRI. Tissue was further sectioned to 100 μm with a microtome. MATLAB was used for further data processing. Segmentation of the internal structures of the brainstem was performed with the BigBrain database. RESULTS Thirteen entry zones were reported and assessed for their safety, including the anterior mesencephalic zone, lateral mesencephalic sulcus, interpeduncular zone, intercollicular region, supratrigeminal zone, peritrigeminal zone, lateral pontine zone, median sulcus, infracollicular zone, supracollicular zone, olivary zone, lateral medullary zone, and anterolateral sulcus. The microanatomy, safety, and approaches are discussed. CONCLUSIONS PLI and 11.7-T MRI data show that a neurosurgeon possibly does not need to consider the microanatomical structures that would not be visible on conventional MRI and tractography when entering the mentioned safe entry zones. However, the detailed anatomical images may help neurosurgeons increase their understanding of the internal architecture of the human brainstem, which in turn could lead to safer neurosurgical intervention.