Using the double immunostaining method, the glia limitans on the surfaces of mouse and monkey brain and spinal cord were studied systematically. The results showed that the superficial glia limitans of mouse brain and spinal cord comprise a layer of surface astrocytes, while the glia limitans of monkey comprise a layer of foot-plates from marginal regions as described in histology and neuroscience textbooks. These surface astrocytes first appear at embryonic day (E)16 in spinal cord and at E17 on the ventral surface of the brain. At postnatal day (P)1, a layer of astrocytes covered the outermost regions of the spinal cord. At P10, the layer of astrocytes also covered the brain surface. The highest proliferation rates of surface astrocytes were found at E17 in the spinal cord and at E18 in the forebrain. Anat Rec, 296:995–1007, 2013. © 2013 Wiley Periodicals, Inc.