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The chubbyhead barb, Enteromius anoplus, as currently described, is the most widely distributed freshwater fish in South Africa. The species occurs in almost all the major river systems across the country, with the exception of the small coastal drainages on the south coast. The use of a comprehensive data set of mitochondrial (mtDNA) cytochrome b (cyt b) sequences uncovered the presence of four distinct lineages or operational taxonomic units (OTUs) within E. anoplus: (a) the Gouritz lineage endemic to the Gouritz River system, (b) the Olifants lineage endemic to the Olifants-Doring River system, (c) the Orange lineage endemic to the Orange River system and (d) the Eastern Cape lineage which is distributed across six river systems from the Great Fish to the Buffalo rivers in the Eastern Cape Province of South Africa. The present study provides a new diagnosis for E. anoplus s.s. (the Gouritz lineage), revalidates Enteromius cernuus (the Olifants lineage) and Enteromius oraniensis (the Orange lineage) as distinct species. The study also provides a description for a new species, Enteromius mandelai sp. nov (the Eastern Cape lineage). E. cernuus and E. mandelai differ from both E. anoplus and E. oraniensis by having a complete lateral line (vs. an incomplete lateral line in the latter species). E. cernuus further differs from the other three species by having long maxillary barbels which reach or exceed the vertical through the middle of the eye and the lowest number of circumpendicular scales (10-12 vs. 12-16 for the other three species). E. oraniensis is distinctive from the other three species by having inconspicuous barbels. These findings add to the growing body of literature that shows that the freshwater fishes of the Cape Fold and adjacent freshwater ecoregions have narrow geographic ranges. This has ramifications for conservation planning and management, as well as the understanding of the evolutionary history of the stream fishes in these global endemic hotspots., (© 2021 Fisheries Society of the British Isles.)