Rat strains differ in physiology, behavior, and recovery after central nervous system injury. To assess these differences, we compared the gross and local anatomy and neuromorphology of the lumbar spinal cord of the Wistar and Dark Agouti (DA) strains. The key findings include (i) distinct spatial relationships between vertebrae and spinal segments in the two strains; (ii) Wistar rats have larger volumes of spinal cord gray and white matter; (iii) DA rats have smaller total neuronal populations, thus indicating an expectation of smaller local neuronal populations; (iv) this expectation was confirmed for interneurons expressing calbindin 28 kDa. But contrary to expectations, (v) DA rats had more numerous populations of the interneurons expressing parvalbumin and a population of α-motoneurons. Consequently, these strains displayed divergent ratios in specific spinal neuronal populations. Researchers should consider these inter-strain differences when comparing data across different strains. © 2024 Wiley Periodicals LLC.