N-methyl-D-aspartate receptors (NMDARs) are glutamatergic receptors that consist of subunit variants GluN2A, GluN2B, and GluN2D. The superficial dorsal horn (SDH) is a critical site for nociceptive signaling; however, the expression of GluN2 subunits in the SDH remains unknown across sex and species. We used immunohistochemistry to identify the expression of GluN2 subunits in the SDH in rat and human adult spinal cord tissue across sex. We identified an enhanced GluN2 subunit expression in the SDH of rats and humans across sexes. GluN2A expression showed diffuse expression in juvenile rats; however, expression becomes localized to the SDH into adulthood. We also found that GluN2 subunits are preferentially expressed in the lateral SDH of adult rats; however, GluN2 subunit expression was uniform across the mediolateral axis in humans. Therefore, NMDAR subunit distribution in the dorsal horn appears to be relatively conserved across species and sex but diverges across postnatal development in rats.