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The periaqueductal gray matter (PAG) is a midbrain structure, involved in key homeostatic neurobiological functions, such as pain modulation and cardiorespiratory control. Animal research has identified four subdivisional columns that differ in both connectivity and function. Until now these findings have not been replicated in humans. This study used high‐resolution brainstem optimized diffusion magnetic resonance imaging and probabilistic tractography to segment the human PAG into four subdivisions, based on voxel connectivity profiles. We identified four distinct subdivisions demonstrating high spatial concordance with the columns of the animal model. The resolution of these subdivisions for individual subjects permitted detailed examination of their structural connectivity without the requirement of an a priori starting location. Interestingly patterns of forebrain connectivity appear to be different to those found …