AbstractThere is a paucity of molecular DNA barcoding informatics on the South African fauna, particularly on terrestrial small mammals. This study tested the utility of DNA barcoding in the dark-footed forest shrew (Myosorex cafer) from forested regions of the Eastern Cape and KwaZulu-Natal provinces of South Africa. Sampled forests included coastal scarp, dune forests and inland Afromontane mistbelt forests. Sequences of mtDNA cytochrome oxidase subunit I (COI, 623 bp), were generated for a total of 78 specimens representing Myosorex cafer (n = 72), Myosorex varius (n = 2), Crocidura cyanea (n = 2) and C . mariquensis (n = 2). Due to the fragmented nature of these forests, we also investigated the cranial morphology of Myosorex cafer, which is strictly confined to forests. Analyses of sequence data produced phylogenetic trees that were consistent with morphological identifications. Genetic data suggest that the movement of these animals between other forest types and the Amatole mistbelt forests has been restricted, as they are too far west of scarp forests to have been recolonized by them. This is the first study that supplies COI sequences of a South African Myosorex species, thus increasing the availability of DNA barcodes of South African small mammals on BOLD.