A real-time PCR technique that allows for accurate and sensitive quantitation of tissue parasite burden in animals infected with the protozoan parasite Trypanosoma cruzi was developed. The utility of this method was demonstrated by confirmation of higher parasite load in mice with acute infections in comparison to chronically infected mice, detection of tissue-restricted parasite persistence in different parasite:host strain combinations, and the observation of increased tissue parasite burden with higher infective doses. This method should be a useful tool for monitoring parasite burden in hosts under various treatment regimens.