Changes in agricultural practices and the resulting extinction of wildlife have led to the reduction or disappearance of savannah tsetse species. Riparian tsetse such as Glossina palpalis gambiensis Vanderplank 1949 and Glossina tachinoides Westwood 1850 (Diptera: Glossinidae) continue to persist in peridomestic sites, transmitting trypanosomiasis. At present, little is known about interspecies differences in feeding behaviour in these two species in southeast Mali, or of the phenomenon of multiple bloodmeals. To study these topics, 279 samples of G. p. gambiensis and G. tachinoides containing host DNA, caught in the Sikasso region between November 2008 and April 2009, were analysed by applying host species-specific primers and sequencing. Human accounted for > 66% of G. p. gambiensis bloodmeals, whereas G. tachinoides contained in equal parts DNA of human, cattle or both, showing a significantly higher proportion of multiple host use. Further, the trypanosome infection rate was found to be three-fold higher in G. tachinoides. Logistic regression analysis revealed double-feeding and infection to be independent of one another, but showed infection to be correlated with engorgement in G. p. gambiensis and female sex in G. tachinoides. Enhanced host-seeking activities paired with the high trypanosome infection rate found in G. tachinoides would indicate that this species has a higher vectorial capacity than G. p. gambiensis.