Barbus pallidus as it is presently defined has a disjunct distribution that is divided between northern and southern populations in South Africa. Sequence data from the cytochrome b gene region showed two distinct lineages that correspond to these geographically distant areas. Divergence between these two lineages was relatively high (5.5–6.5%) and comparable to typical interspecific divergences found between closely related southern African cyprinid fishes. Based on this deep genetic divergence, we conclude that the northern lineage may represent a previously unrecognised species or may belong to a different, but known species or species complex not associated with ‘true’ B. pallidus. These findings add to a growing body of evidence that freshwater fish diversity in southern Africa requires major revision.