NMDARs are excitatory glutamate receptors expressed in the superficial dorsal horn (SDH) of the spinal cord. The GluN2 subtypes of NMDAR subunit confer NMDARs with structural and functional variability, enabling heterogeneity in synaptic transmission and plasticity. Surprisingly, there is a complete lack of knowledge on GluN2 expression in female rodent or human spinal cord. In this study we aimed to investigate the relative expression of specific GluN2 variants in the lumbar SDH of both male and female rats and humans. We found a dominant expression of GluN2B and GluN2D in the SDH of male rats, while only GluN2B was preferentially localized to the SDH of females. GluN2B subtype was also more abundantly expressed in the medial compared to the lateral SDH, in male rats only. Finally, we adapted the staining approaches from rodents to human spinal tissue for future investigations on whether GluN2 subtypes expression patterns are conserved in humans.