We adopted an integrated systematic approach to delimit evolutionary species and describe phylogeographic, morphometric and ecological relationships in Otomys denti (from the Albertine Rift, Southern Rift in Malawi and the northern Eastern Arc Mountains) and Otomys lacustris (from the Southern Rift in Tanzania and Zambia, and the southern Eastern Arc Mountains). Molecular [cytochrome (cyt) b sequences, 1143 bp, N = 18], craniometric (classical, N = 100 and geometric, N = 60) and ecological (Partial Least Squares regression of shape and ecogeo- graphic variables) approaches show a profound, parallel disjunction between two groups: (1) Eastern Arc and Southern Rift (including the Malawi Rift) (O. lacustris and Otomys denti sungae) and (2) Albertine Rift (Otomys denti denti and Otomys denti kempi) taxa. Within both groups, cyt b sequences or craniometric analysis provided evidence for the differentiation of both southern and northern Eastern Arc from Southern Rift lineages (across the so-called Makambako Gap). Within the Albertine Rift (denti–kempi) lineage, populations from individual mountain ranges differed significantly in skull shape (but not size), but were similar genetically. Over-reliance in the past on very few morphological characters (e.g. number of molar laminae) and a polytypic species concept has obscured phylogenetic relationships and species discrimination in this group. We recognize at least three species in this group, and distinct lineages within two of these species. Each species or lineage was endemic to one of three regions: the Albertine Rift, the Malawi Rift or the Eastern Arc. Our result echo conclusions of recent studies of other mammalian and bird taxa and reflect the geomorphology and palaeoclimatic history of the region.