The human serum resistance associated (SRA) gene isolated from a Ugandan strain of Trypanosoma brucei rhodesiense has been shown to be capable by itself of conferring the trait of human infectivity on T.b. brucei by transfection. This gene has also been identified in several other isolates of T.b. rhodesiense, but not in the other human pathogenic trypanosome in Africa, T.b. gambiense, casting doubt on its ubiquity and function. Here, we show that this gene occurs in T.b. rhodesiense from sleeping sickness foci throughout East Africa (Ethiopia, Uganda, Kenya, Tanzania, Zambia, Botswana), but is not found in T.b. brucei isolates or any other trypanosomes of subgenus Trypanozoon. SRA genes from 10 T.b. rhodesiense isolates from five disease foci were compared and were 97.9-99.7% homologous, with three minor sequence variants. PCR amplification of this gene forms the basis of a new test to identify T.b. rhodesiense. This is the first molecular marker identified for T.b. rhodesiense, despite intensive efforts over the past 20 years. It will be invaluable for identification of animal reservoir hosts and detection of T.b. rhodesiense in its tsetse fly vector. Strain typing using minisatellite markers showed considerable genetic heterogeneity between T.b. rhodesiense isolates, despite the presence of the conserved SRA gene. These results are consistent with the hypothesis that new T.b. rhodesiense strains arise by genetic exchange among T. brucei ssp. spreading the SRA gene and thereby the trait for human serum resistance and human infectivity.