The anatomical connections of the pedunculopontine nucleus (PPN), a brainstem structure associated with locomotion, have been determined recently in healthy humans using probabilistic diffusion tractography (PDT). In order to compare these with histologically demonstrated connections of the PPN in monkeys, and thus to support the use of PDT in humans, we have carried out PDT in a fixed rhesus monkey (Macaca mulatta) brain. Probabilistic diffusion tractography was carried out in a fixed post-mortem rhesus monkey brain using diffusion data acquired at 3T MRI (60 directions × 5 averages, b = 3000 s/mm2, matrix size = 104 × 132 × 96, 720 × 720 × 720 μm voxels). We identified the major connections of the PPN from single seed voxels that could be confidently located within the nucleus on the diffusion images. The organisation of these connections within a 3 × 3 × 3 voxel (∼10 mm3) region surrounding the initial seed voxel was then examined. PDT confirmed that the rhesus monkey PPN connections with the basal ganglia and motor cortical areas matched those previously demonstrated using conventional anatomical tracing techniques. Furthermore, although the organisation of subcortical connections within the PPN has not been extensively demonstrated in animals, we show here in a rhesus monkey that there are clearly separated connections of the PPN with the thalamus, substantia nigra, and subthalamic nucleus. Thus, in addition to increasing confidence in the accuracy of PDT for tracing PPN connections and determining the organisation of these connections within the PPN in vivo, our observations suggest that diffusion tractography will be a useful new technique to rapidly identify connections in animal brains pre-mortem and post-mortem.