Chromosomal polytypy, morphological conservatism and absence of data have frustrated the taxonomic revision of two species of southern African-endemic laminate-toothed rats (Otomys irroratus and Otomys saundersiae s.l.). New cytogenetic (G-banding and fluorescence in situ hybridization), DNA sequence [cytochrome b (cyt b) gene] and geometric morphometric data demonstrate the synonymy of O. saundersiae from Grahamstown (Eastern Cape, South Africa) under O. irroratus, and the validity of Otomys karoensis from the Fynbos Biome of the Western Cape. Phenotypic dimorphism in pelage colour and cranial morphology in O. irroratus from the climatically unpredictable Albany Thicket (=Savanna) Biome of the Eastern Cape results from the retention of allometric paedomorphic traits in some adults (saundersiae morph) but not others. The same paedomorphic traits are associated with speciation and karyotypic and genetic differentiation in O. karoensis. Within O. irroratus, two phenotypically and genotypically (cyt b divergence=6.4%) divergent lineages correspond with the Fynbos/Albany Thicket and Grassland biomes. Incipient speciation in O. irroratus seems to be associated with ecology rather than karyotype.