The medial temporal lobe (MTL) plays a key role in learning, memory, spatial navigation, emotion, and social behavior. The improvement of noninvasive neuroimaging techniques, especially magnetic resonance imaging, has increased the knowledge about this region and its involvement in cognitive functions and behavior in healthy subjects and in patients with various neuropsychiatric and neurodegenerative disorders. However, cytoarchitectonic boundaries are not visible on magnetic resonance images (MRI), which makes it difficult to identify precisely the different parts of the MTL (hippocampus, amygdala, temporopolar, perirhinal, entorhinal, and posterior parahippocampal cortices) with imaging techniques, and thus to determine their involvement in normal and pathological functions. Our aim in this study was to define neuroanatomical landmarks visible on MRI, which can facilitate the examination of this region. We examined the boundaries of the MTL regions in 50 post-mortem brains. In eight cases, we also obtained post-mortem MRI on which the MTL boundaries were compared with histological examination before applying them to 26 in vivo MRI of healthy adults. We then defined the most relevant neuroanatomical landmarks that set the rostro-caudal limits of the MTL structures, and we describe a protocol to identify each of these structures on coronal T1-weighted MRI. This will help the structural and functional imaging investigations of the MTL in various neuropsychiatric and neurodegenerative disorders affecting this region. Hum Brain Mapp 35:248–256, 2014. © 2012 Wiley Periodicals, Inc.