Trypanosoma rangeli can infect humans and the same domestic and wild animals and triatomine vectors infected by T. cruzi in Central and South America. This overlapping distribution complicates the epidemiology of Chagas disease because of the cross-reactivity between T. rangeli and T. cruzi antigens. We have studied T. rangeli strains isolated from different geographical regions using the mini-exon gene as a genetic marker. Two pairs of oligonucleotides directed to this gene were designed in order to detect specifically T. rangeli DNA by PCR assays. This assay was highly sensitive, able to amplify the target sequence using the equivalent DNA content of a single parasite as template, and demonstrated no cross-reactivity with T. cruzi DNA. T. rangeli SC-58 strain, isolated in southern Brazil, showed a distinct electrophoretic pattern from the other T. rangeli strains tested. Low stringency single specific primer-PCR (LSSP) assays were able to detect sequence polymorphisms at the mini-exon gene among T. rangeli strains. Sequence comparisons of this gene revealed that the SC-58 strain was genetically distinct from strains isolated in Central America and northern South America. In addition to insertion/deletion events, the presence of microsatellite repeats in the non-transcribed region of the gene contribute to the intra-species variability.