Phylogenetic relationships of Sciurus were examined based on the mitochondrial cytochrome b gene sequences of three Old World and four New World species. The purpose was to test for monophyly in Old World Sciurus (S. anomalus, S. lis, and S. vulgaris). Phylogenetic trees well supported clustering of (1) S. anomalus, (2) S. lis and S. vulgaris, and (3) the four New World Sciurus species. Sciurus anomalus was more closely related to the clade consisting of New World Sciurus species than the one consisting of S. lis and S. vulgaris, indicating a polyphyletic relationship in Old World Sciurus. The primary divergence of Sciurus may have taken place early on the Eurasian Continent. Then, the ancestral stock of modern New World Sciurus would have migrated from the Eurasian Continent to the North American Continent.