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Abstract.  Reciprocal-crossing experiments were carried out and mitochondrial cytochrome oxidase I gene (mtCOI) sequences were compared for allopatric and sympatric Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) populations collected from Africa and India, and from the host-plants cassava, sweet-potato and a common weed, Euphorbia geniculata. Three incompatible mating groups were discovered, which involved the cassava B. tabaci colonies from Africa and India, the cassava and sweet-potato B. tabaci populations from Uganda, and the cassava and E. geniculata B. tabaci from India. Successful reciprocal mating occurred between cassava-specific B. tabaci from Uganda, Tanzania and Ghana, and between two Indian cassava B. tabaci populations. The parsimony and neighbour-joining analyses of 699 bp mtCOI gene sequences divided the colonies primarily into those originating from Africa and India. Further subgrouping corresponded to host-plant specialization. Cassava-specific Ugandan, Tanzanian and Ghanaian colonies formed a single group and the sympatric sweet-potato colony from Uganda grouped separately from them. The two geographically distant Indian cassava B. tabaci populations were similar and formed a single group, whereas the sympatric E. geniculata colony formed a sister clade. The clades generated by the phylogenetic analyses were maintained, with highly supported bootstrap values, when other published mtCOI gene sequences were included in the tree-building process and the divisions matched those revealed by the reciprocal-crossing experiments. These data suggest that biologically discrete populations exist within B. tabaci (sensuRussell, 1957).