The karyotypes of six species of Pseudobarbus Smith, 1841, namely P. afer (Peters, 1864), P. asper (Boulenger, 1911), P. burchelli Smith, 1841, P. burgi (Boulenger, 1911), P. phlegethon (Barnard, 1938) and P. tenuis (Barnard, 1938), were examined by conventional Giemsa staining and described. All six karyotypes have 2n = 100 chromosomes, dominated by bi-armed chromosomes, as does the only other member of the genus, P. quathlambae (Barnard, 1938). Sex-related intraspecific karyotype variation was not found. The shared chromosome numbers and general similarity of the karyotypes (FN = 186–192) provide a new synapomorphy to support their monophyly, which is already indicated by anatomical and mtDNA markers. Karyotype evolution within the genus has been accompanied by chromosomal inversions and centromeric shifts. Comparison of the diploid number found in Pseudobarbus with other African barbine cyprinines, which have in the region of 2n = 50 and lower FNs, suggests a tetraploid evolutionary origin of the genus, possibly by allotetraploidy.