We present an extensive DNA barcoding study of the fish species of the Lake Edward system, including information on intraspecific variation. The DNA barcode gene, cytochrome c oxidase I (COI), failed to discriminate the 35 species of the genus Haplochromis analysed. For the non-Haplochromis species, the reference library had a near complete coverage of 91.2%, with 31 out of the 34 known species of the system sequenced. With the recent morphology-based review of the ichthyofauna of the system as a backbone, the DNA barcoding library proved to be very effective for future species identifications. High identification successes were obtained based on Best Match (98.2%), Best Close Match (97.9%), and All Species Barcode (95.0%) criteria. Surprisingly, Laciris pelagicus and Micropanchax vitschumbaensis, two morphologically distinct species, had very similar and even identical COI haplotypes, whilst in Enteromius the existence of possible cryptic species was revealed. Few species occurring in the Lake Edward system were already represented in the global database Genbank. For some widespread species, regional genetic differences were found, highlighting the value of basin-specific information in reference databases. The provided reference library of DNA barcodes is a valuable tool for future conservation actions, which is certainly relevant in a system where the fisheries are under pressure.