Neuroimaging methods have so far identified various structures in the brain involved in the processing of pain and its control. However, our understanding of their anatomical connectivities is relatively weak. Diffusion tensor imaging (DTI), a magnetic resonance imaging-based method, allows in vivo mapping of the anatomical connections in the human brain and was used to investigate the white matter connections originating from the periaquaductal grey (PAG) and nucleus cuneiformis (NCF). We performed DTI on 8 healthy right-handed male volunteers. Group analysis showed that tract paths could be defined and their likelihood quantified for connections between the PAG and separately for the NCF, to the prefrontal cortex, amygdala, thalamus, hypothalamus and rostroventral medial medulla bilaterally. The connections identified confirm the existence of an anatomical circuitry for the functionally characterised top-down influences on pain processing via brainstem structures in humans.