Preclinical and clinical evidence suggests that cannabis, a potent cannabinoid, has potential analgesic properties. However, there is a gap in the literature in respect to cannabinoid receptor expression and localization in the spinal cord across both sex and species, with almost nothing known in humans. We used immunohistochemistry and confocal imaging to investigate the differential expression of the cannabinoid type 1 receptor (CB1R) across dorsal horn laminae and cell populations in male and female adult rats and humans. Qualitatively, we observed increased neuropil immunostaining in the SDH of rats and humans, and somatic staining in deeper laminae. Quantitative results indicated a significant increase in CB1R immunoreactivity in the SDH when compared to the deeper dorsal horn laminae of male and female rat and humans. The preferential expression of CB1Rs in the SDH across both sex and species has significant implications for both the understanding and treatment of pain.