Species of Alestidae are known to be parasitised by dactylogyrid monogeneans representing three genera, Afrocleidodiscus Paperna, 1969, Annulotrema Paperna & Thurston, 1969, and Characidotrema Paperna & Thurston, 1968. The objective of the present study was to investigate the species diversity of Monopisthocotylea of African tetras from the Lower Phongolo River and floodplain in South Africa. Four new and two previously described species of Annulotrema were identified from the gills of three species of African tetras, Brycinus imberi, Hydrocynus vittatus, and Micralestes acutidens. The collected parasites were studied using two complementary approaches: morphology of hard sclerotised structures, and molecular markers using rDNA sequence data (28S rDNA, 18S rDNA, and ITS1). Three new species, Annulotrema arcum n. sp., Annulotrema caputfemoris n. sp., and Annulotrema strepsiceros n. sp., were described from B. imberi and one species, Annulotrema retortum n. sp., from M. acuditens. Two previously described species, Annulotrema pikoides Guégan, Lambert and Birgi, 1988 and A. pseudonili Kičinjaová and Řehulková, 2017, were newly recorded from H. vittatus in South Africa. Annulotrema arcum n. sp. and A. caputfemoris n. sp. share similar male copulatory organ morphologies, suggesting a close phylogenetic relationship as sister taxa. Despite weak nodal support, A. strepsiceros n. sp. shows morphological congruence with the former two species, reinforcing their molecular linkage. The present study shows a critical need for the exploration of monopisthocotylean diversity and the paucity of available molecular data of representatives from this group.