The Nile Delta and Valley act as an important natural ecological barrier that the animals of the Western and Eastern Deserts cannot cross. For historical biogeographic reconstructions of arid habitats, the genus Acomys is an excellent model. Barriers that restrict the movement of animals allow for geographic isolation. In the present study, to investigate the evolutionary forces driving the differentiation of the Cairo spiny mice, Acomys cahirinus, particularly as related to the biogeographical relationship between the Western and Eastern Deserts of Egypt, thirty-eight skulls of male specimens of the Cairo spiny mouse from four different regions of Egypt that were kept in Al-Azhar University Zoological Collection in Cairo, Egypt were examined. Five of these skulls came from Bahariya Oasis, seven from Dakhla Oasis, five from Helwan region, and twenty-one came from Eastern Desert. Based on sixteen absolute cranial and dental measurements and seventeen ratios used here, A. c. helmyi from Dakhla Oasis differ greatly from those from Bahariya Oasis (A. c. cahirinus), Helwan region (A. c. cahirinus), and Eastern Desert (A. c. hunteri) populations. There were many cranial and dental characteristics correlated with each other. Most of these characteristics are highly positively correlated with body mass, condyloincisive length, and mastoid width. Despite the presence of this natural ecological barrier (the Nile Delta and Valley), a great similarity was found between the Cairo spiny mouse populations from Bahariya Oasis (Western Desert) and from Eastern Desert, noting the great difference between the Cairo spiny mouse populations from Dakhla Oasis (Western Desert) from the other populations in the present study.