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The present study was carried out to spot fairly the limit of tsetse fly belt in river Khor Yabus, from Sudan-Ethiopian border in the Blue Nile State,Sudan as the successive ecological changes might create significant modification in tsetse distribution. Moreover to draw attention to circulating Trypanozoon species via detecting infection on tsetse flies using DNA-PCR technique.The results obtained showed that only 8 flies out of 180 examined (4.44%) harboring Trypanosoma brucei group as they reacted positively with TBR1&2 primers, out of these only 3 flies (37.5%) reacted positively with SRAA&E primers, hence were considered infected with T. b. rhodesiense. However, none of the examined flies were found infected with T. b. gambiense. Thus the burden of flies harboring T. b. rhodisense and T.b. brucei trypanosomes were 1.67% and 2.78%, respectively. This is first ever record of the presence of T. b. rhodesiense in the Yabus district.The above results are discussed in relation to the possibility of conducting active sleeping sickness surveillance and tsetse fly control campaigns preceding the land use intended to take place in the region.