Summary Objective To determine the drug resistance of Trypanosoma brucei rhodesiense strains isolated from sleeping sickness patients in Tanzania. Method We first screened 35 T. b. rhodesiense strains in the mouse model, for sensitivity to melarsoprol (1.8, 3.6 and 7.2 mg/kg), diminazene aceturate (3.5, 7 and 14 mg/kg), suramin (5, 10 and 20 mg/kg) and isometamidium (0.1, 1.0 and 2 mg/kg). A 13 isolates suspected to be resistant were selected for further testing in vitro and in vivo. From the in vitro testing, IC50 values were determined by short-term viability assay, and MIC values were calculated by long-term viability assay. For in vivo testing, doses higher than those in the initial screening test were used. Results Two T. b rhodesiense stocks expressed resistance in vivo to melarsoprol at 5 mg/kg and at 10 mg/kg. These strains had high IC50 and MIC values consistent with those of the melarsoprol-resistant reference strain. Another isolate relapsed after treatment with 5 mg/kg of melarsoprol although it did not appear resistant in vitro. One isolate was resistant to diminazene at 14 mg/kg and another was resistant at both 14 and 28 mg/kg of diminazene. These two isolates had high IC50 values consistent with the diminazene-resistant reference strain. Two isolates relapsed at a dose of 5 mg/kg of suramin, although no isolate appeared resistant in the in vitro tests. Two isolates were resistant to isometamidium at 1.0 mg/kg and had higher IC50 values. Two isolates were cross-resistant to melarsoprol and diminazene and one isolate was cross-resistant to suramin and isometamidium. Conclusion The reduced susceptibility of T. b. rhodesiense isolates to these drugs strongly indicates that drug resistance may be emerging in north–western Tanzania.