•COI can delineate species of the Aftrotropical Atylotus genus.•Many of the Afrotropcial Atylotus records in BOLD are incorrectly identified.•The status of Atylotus fuscipes is revived to species. Previous studies of the horse fly genus Atylotus in the Afrotropics has shown little to no differentiation into species based on the barcoding region of cytochrome oxidase I (COI), largely due to morphological misidentifications. Using field caught specimens and a museum reference collection together with type comparisons, COI and 16S ribosomal RNA sequences were generated from two specimens of Atylotus agrestis, two A. albipalpus, four A fuscipes and one A. nigromaculatus. Phylogenetic analysis of these sequences produced four separate species clades with strong support. The results showed that COI does delineate the species of Afrotropical Atylotus and that misidentifications of specimens is a common problem. Additionally, Atylotus fuscipes is revived from synonymy and given full species status. Finally, a comprehensive review of the COI barcodes, publicly available on GenBank and BOLD is included that highlights some problems with using sequences from public databases.