As part of the feasibility study for an onchocerciasis vector elimination project we investigated the isolation of the Tukuyu focus in Tanzania from possible vector re-invasion. This was achieved by examining the distribution of the Simulium damnosum complex vector cytospecies outside the focus to look for potential sources of re-invasion. Besides cytotaxonomic identifications of the aquatic stages, we applied morphotaxonomic and molecular techniques to identify S. thyolense and confirm it as the anthropophilic species in both the Tukuyu and the neighbouring Ruvuma foci. We detected significant differences in chromosome inversion frequencies between the Tukuyu populations and those breeding to the southwest in the adjacent Songwe river basin and in northern Malawi (where there is no man-biting and no onchocerciasis), suggesting that there is not normally a great deal of migration in either direction. By contrast, populations of S. thyolense from the Tukuyu and Ruvuma foci (150km southeast of Tukuyu) were much more similar in terms of their chromosomal polymorphisms, indicating a higher possibility of re-invasion, although migration is still restricted to some extent, as indicated by some differences in chromosome polymorphisms between the two foci. Future migratory events which might be associated with vector control operations can be monitored by vector cytospecies identification, the frequency of polymorphic inversions which characterise the different vector populations, and the identification of accompanying non-vector cytospecies (e.g. S. plumbeum and cytotype Kasyabone occur exclusively in the two foci, and hence their re-appearance in Tukuyu could have only one outside source). The morphology of the scutal pattern of neonate males may act as a quick test for vector species identification where chromosome squashes are unavailable.