Intraspecific phylogenies can provide useful insights into how populations have been shaped by historical and contemporary processes. Taiwan formed around 5 million years ago from tectonic uplift, and has been connected to mainland Asia several times since its emergence. A central mountain range runs north to south, bisecting the island, and potentially impedes gene flow along an east–west axis. The Formosan lesser horseshoe bat (Rhinolophus monoceros) is endemic to Taiwan, where it is found mainly at low altitude. To determine the population structure and the demographic and colonization history of this species, we examined variation in the mitochondrial DNA control region in 203 bats sampled at 26 sites. We found very high haplotype and nucleotide diversity, which decreased from the centre to the south and north. Population differentiation followed a pattern of isolation by distance, though most regional genetic variance was attributable to differences between the relatively isolated southern population and those from other regions. A haplotype network was consistent with these findings and also suggested a southward colonization, followed by subsequent secondary contact between the south and other regions. Mismatch distributions were used to infer a past population expansion predating the last glacial maximum, and a neighbour-joining tree showed that R. monoceros formed a monophyletic grouping with respect to its sister taxa. Taken together, our results suggest that this taxon arose from a single period of colonization, and that demographic growth followed in the late Pleistocene. Current genetic structure reflects limited gene flow, probably coupled with stepwise colonization in the past. We consider explanations for the persistence of the species through multiple glacial maxima.