Lake Tana, located in the north-western highlands of Ethiopia, contains a unique assemblage of cyprinid fishes. In addition to the only known intact species flock of large (max. 100 cm forklength (FL)) Labeobarbus species, the lake harbours three small (<10 cm FL) Barbus species: B. humilis Boulenger, 1902, B. pleurogramma Boulenger, 1902 and B. tanapelagius de Graaf, Dejen, Sibbing and Osse, 2000. Phylogenetic relationships of the small Barbus species of Lake Tana were investigated using the mtDNA cytochrome b gene. All small Barbus species occurring in Lake Tana are part of the 'small African diploid' groups identified by Tsigenopoulos et al. (2002). Barbus pleurogramma, only reported in Lake Tana, and populations of the morphologically similar species, B. paludinosus Peters, 1852, collected in rivers and lakes throughout Ethiopia, comprised a monophyletic group with separate clades congruent with drainage basins. Barbus pleurogramma could well be a cryptic species rather than synonymous to B. paludinosus as previously suggested. The genetic divergence between B. humilis and B. tanapelagius was very low, and without lineage sorting of haplotypes. This is probably due to recent (<16 000 years ago) evolution of the pelagic, zooplanktivorous B. tanapelagius from the benthic, omnivorous B. humilis after the formation of Lake Tana.