The African pygmy mice (Mus, subgenusNannomys) are a group of small-sized rodents that occur widely throughout sub-Saharan Africa. Chromosomal diversity within this group is extensive and numerous studies have shown the karyotype to bea useful taxonomic marker. This is pertinent toMus minutoidespopulations in South Africa where two different cytotypes(2n = 34, 2n = 18) and a modification of the sex determination system (due to the presence of a Y chromosome in somefemales) have been recorded. This chromosomal diversity is mirrored by mitochondrial DNA sequences that unambiguouslydiscriminate among the various pygmy mouse species and, importantly, the differentM.minutoidescytotypes. However, thegeographic delimitation and taxonomy of pygmy mice populations in South Africa is poorly understood. To address this,tissue samples ofM.minutoideswere taken and analysed from specimens housed in six South African museum collections.Partial cytochrome b sequences (400 pb) were successfully amplified from 44% of the 154 samples processed. Two specieswere identified:M. indutusandM. minutoides. The sequences of theM. indutussamples provided two unexpected features: i)nuclear copies of the cytochrome b gene were detected in many specimens, and ii) the range of this species was found toextend considerably further south than is presently understood. The phylogenetic analysis of theM. minutoidessamplesrevealed two well-supported clades: a Southern clade which included the two chromosomal groups previously identified inSouth Africa, and an Eastern clade that extended from Eastern Africa into South Africa. Congruent molecular phylogeneticand chromosomal datasets permitted the tentative chromosomal assignments of museum specimens within the differentclades as well as the correction of misidentified museum specimens.