Full text can be accessed at http://www.sciencedirect.com/science/article/pii/S156713481300110XDiplostomid trematodes comprise a large and diverse group of widespread digeneans whose larval stages are important parasitic pathogens that may exert serious impacts in wild and cultured freshwater fish. However, our understanding of their diversity remains incomplete especially in the tropics. Our study is the first application of a DNA-based approach to diplostomid diversity in the African continent by generating a database linking sequences for the mitochondrial cytochrome c oxidase subunit 1 (cox1) barcode region and ITS1-5.8S-ITS2 rRNA gene cluster for brain-infecting diplostomid metacercariae from the catfish Clarias gariepinus. Analyses of newly-generated partial cox1 sequences for 34 larval isolates of Tylodelphys spp. from Tanzania and Diplostomum spp. from Tanzania and Nigeria revealed three strongly supported reciprocally monophyletic lineages of Tylodelphys spp. and one of an unknown species of Diplostomum. The average intraspecific divergence for the cox1 sequences for each recognised novel lineage was distinctly lower compared with interspecific divergence (0.46–0.75% vs 11.7–14.8%). The phylogenetic hypotheses estimated from Bayesian inference and maximum likelihood analyses of ITS1-5.8S-ITS2 data exhibited congruent strong support for the cox1-derived lineages. Our study thus provides molecular-based evidence for the existence of three distinct brain-infecting species co-occurring in natural populations of C. gariepinus. Based on phylogenetic analyses, we re-allocated Diplostomum mashonense Beverley-Burton (1963) to the genus Tylodelphys as a new combination. We also generated cox1 and ITS1-5.8S-ITS2 sequences for an unknown species of Diplostomum from another African fish host, Synodontis nigrita.