Dactylogyrus Diesing, 1850 is the most species-rich genus in Platyhelminthes, with over 900 documented species, that are mostly strictly specific to freshwater cyprinoids. The morphological Dactylogyrus groups afrobarbae-type, carpathicus-type, pseudoanchoratus-type, and varicorhini-type are known to occur in Africa. This study describes a new species of Dactylogyrus of the varicorhini-type from the gills of the endemic smallmouth yellowfish Labeobarbus aeneus (Burchell) from the Vaal River, Free State Province, South Africa. Dactylogyrus matlopong sp. n. is unique among its varicorhini-type congeners mainly by the accessory piece of the male copulatory complex that presents a hook-shaped subunit with defined round base not reaching the male copulatory organ, combined with the presence of a conspicuous medial projection on the anterior margin of the ventral bar. Sequences of the partial 28S and 18S rRNA genes, together with entire ITS1 region, were generated for the first time for a species of Dactylogyrus from South Africa. Concatenated phylogenetic analyses of selected Dactylogyrus spp. showed that these parasites group according to their morphological types.