The hypothesis of Pleistocene forest refugia was tested using comparative phylogeography of Scotonycterini, a fruit bat tribe endemic to Africa containing four species: Scotonycteris zenkeri, Casinycteris argynnis, C. campomaanensis, and C. ophiodon. Patterns of genetic structure were assessed using 105 Scotonycterini (including material from three holotypes) collected at 37 localities, and DNA sequences from the mitochondrial cytochrome b gene (1140 nt) and 12 nuclear introns (9641 nt). Phylogenetic trees and molecular dating were inferred by Bayesian methods. Multilocus analyses were performed using supermatrix, SuperTRI, and *BEAST approaches. Mitochondrial analyses reveal strong phylogeographical structure in Scotonycteris, with four divergent haplogroups (4.9–8.7%), from Upper Guinea, Cameroon, western Equatorial Africa, and eastern Democratic Republic of the Congo (DRC). In C. argynnis, we identify two mtDNA haplogroups corresponding to western and eastern Equatorial Africa (1.4–2.1%). In C. ophiodon, the mtDNA haplotypes from Cameroon and Ivory Coast differ by only 1.3%. Nuclear analyses confirm the validity of the recently described C. campomaanensis and indicate that western and eastern populations of C. argynnis are not fully isolated. All mtDNA clusters detected in Scotonycteris are found to be monophyletic based on the nuclear dataset, except in eastern DRC. In the nuclear tree, the clade from western Equatorial Africa is closely related to individuals from eastern DRC, whereas in the mitochondrial tree it appears to be the sister-group of the Cameroon clade. Migrate-n analyses support gene flow from western Equatorial Africa to eastern DRC. Molecular dating indicates that Pleistocene forest refugia have played an important role in shaping the evolution of Scotonycterini, with two phases of allopatric speciation at approximately 2.7 and 1.6 Mya, resulting from isolation in three main forest areas corresponding to Upper Guinea, Cameroon, and Equatorial Africa. Two cryptic species and two subspecies are described herein in the genus Scotonycteris. Female philopatry and male biased dispersal are supported for the smallest taxa, i.e., the three species of Scotonycteris and C. argynnis. The Congo, Ntem, and Sanaga rivers are identified as biogeographic barriers to the dispersal of Scotonycteris during interglacial periods. A greater capacity for long-distance dispersal is inferred for the largest species, C. ophiodon.