Central America is an ideal region in which to study patterns of historical divergence and population genetic differentiation, because of its extraordinarily dynamic biogeographical, tectonic, and climatic history. The rodent Ototylomys phyllotis is the only extant species of the genus Ototylomys and is distributed within this region from the Isthmus of Tehuantepec, Mexico, to central Costa Rica, offering an excellent opportunity to study spatial and temporal patterns of population structure of the species and to explain the ecological and evolutionary processes responsible for those patterns. We estimated the genetic diversity and structure within and between populations of O. phyllotis, times of divergence, and migration patterns using mitochondrial DNA and a comprehensive combination of phylogenetic and phylogeographical computational analyses. Our results support monophyly of the genus Ototylomys. We identified three major phylogeographical lineages within O. phyllotis that are linked to its diversification and coincide with the main geological features that shaped Middle America. The origin of the genus was before 3.35 Mya, prior to the Great American Biotic Interchange (GABI), and its initial occurrence was near the centre of its current distribution (Honduras/El Salvador), from which it later spread (3.20–2.84 Mya) following a series of GABI pulses. The species showed an initial northward dispersal to the Chiapas and Guatemala highlands (2.27 Mya) followed by diversification. A later dispersal (1.82 Mya) occurred toward both the south (Nicaragua, Costa Rica) and the north (Belize). The Yucatan peninsula was colonized (0.8 Mya) by individuals from Belize. Extremely high radiation and range expansion occurred throughout the entire range, the highest of which was in the Yucatan peninsula (0.125 Mya). © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107, 593–610.