Morphological characters have historically been used as the basis for mammalian taxonomic designations and, in a geographic context, subspecies descriptions. Geographic genetic structuring of a species, however, often reflects a contrasting classification for sampled populations. To investigate morphological and genetic congruence, geometric morphometrics and phylogeographic mitochondrial DNA sequence analyses of a South African plains-dwelling species, Myotomys unisulcatus, the Karoo bush rat, was performed across its range. A Bayesian population structure analysis identified two closely-related distinct genetic assemblages: the first contains populations from both the eastern, southern, and western parts of the species range (coastal lowland group), and the second comprises individuals from the Little Karoo (central interior group). Areas of sharp elevation (the Great Escarpment), coupled to vegetational differences, appeared to be the main factor limiting gene flow between these two groups. Geometric morphometric analyses on the ventral and dorsal views of the crania of M. unisulcatus failed to support the genetic groupings. Instead environmental factors in the respective biomes appeared to play a more important role in shaping the crania of both genders. The contrasting patterns obtained between morphology and genetics in M. unisulcatus is probably indicative of phenotypic plasticity throughout the range of the species, and it is hypothesized that regional environmental factors play a prominent role in explaining geographic morphological variation within the species. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102, 510–526.