The sibling species of the Simulium damnosum complex that are potential vectors in the foci of human onchocerciasis found in southern Malawi (the southern-most foci in Africa) were identified, using a combination of cytotaxonomic and molecular techniques. Simulium thyolense was found to be the most abundant species within the foci (representing 91% of the larvae collected), whilst S. kilibanum and cytoforms of the 'Ketaketa' subcomplex were the dominant members of the complex found outside of the foci. As all 188 biting female flies collected from the Thyolo, Mwanza and Mulanje foci were identified as S. thyolense, this species is probably the only significant vector of Onchocerca volvulus in the area, and the other Simulium species collected are assumed to be zoophilic. The results confirm the diagnostic value of the size of the amplicon(s) produced in PCR based on the sequences coding for the internal-transcribed-spacer region of the nuclear ribosomal DNA (ITS-1 rDNA), with a characteristic 340bp amplicon for S. thyolense. Chromosomal polymorphisms within S. thyolense indicate some degree of geographical isolation of the 'Thyolo-Mulanje' focus from the Mwanza focus.