Although Trypanosoma rangeli is harmless for humans, it is a serious problem since it may be confused with diagnosis of Trypanosoma cruzi, the etiologic agent of Chagas disease. Both parasites overlap geographically, share antigenic protein, and are able to infect the same Triatominae vector and vertebrate host, including human. Our objective was to differentiate T. cruzi and T. rangeli isolates from Colombia based on polymerase chain reaction (PCR) amplification of the minicircles followed by appropriate hybridization tests with selected DNA probes and restriction fragment length polymorphism (RFLP) analysis. We worked with highly characterized T cruzi and T. rangeli isolates from different biologic origins and geographic areas of Colombia, and they were analyzed by RFLP and PCR amplification of variable region of minicircles and Southern blot analysis. Our results and experimental conditions demonstrate the usefulness of PCR amplification of the minicircles followed by Southern blot analysis to differentiate T. cruzi from T. rangeli, which can be highly important to improve diagnosis of Chagas disease. (C) 2010 Elsevier Inc. All rights reserved.