The phylogenetic relationships among “Barbus” species (Barbus jacksonii, B. paludinosus and B. trimaculatus) from Malagarasi and Pangani River systems were examined by partial mitochondrial cytochrome b (cyt.b) gene. Sequences of the same samples from elsewhere in Africa obtained from the South African Institute of Aquatic Biodiversity (SAIAB’s) collection were also included in the analysis. Molecular technique was necessary due to phenotypic plasticity and extensive overlap of morphological features among the “Barbus species”. The findings of this study revealed that B. paludinosus from hale and Pangani populations occurred in two distinct genetic lineages though separated by a distance of approximately 10 km apart. Similar results were also noted amongst the population of B. paludinosus in Songati and Muumbara located on Lake Sagara in Malagarasi River basin. B. trimaculatus and B. jacksonii were indistinguishable such that it is envisaged that the two species are one and the same. The Barbus spp. from Africa included in the current study showed genetic affinity to neither European Barbus spp. nor South African redfins of the Pseudobarbus to conclude that the studied species of barbs are taxonomically quite different that needs further investigation to warrant renaming.