Background: Mitochondrial DNA is a valuable taxonomic marker due to its relatively fast rate of evolution. In Trypanosoma cruzi, the causative agent of Chagas disease, the mitochondrial genome has a unique structural organization consisting of 20-50 maxicircles (∼20kb) and thousands of minicircles (0.5-10kb). T. cruzi is an early diverging protist displaying remarkable genetic heterogeneity and is recognized as a complex of six discrete typing units (DTUs). The majority of infected humans are asymptomatic for life while 30-35% develop fatal cardiac and/or digestive syndromes. However, the relationship between specific clinical outcomes and T. cruzi genotype remains elusive. The availability of whole genome sequences has driven advances in high resolution genotyping techniques and re-invigorated interest in exploring the cryptic genetic diversity present within the various DTUs. Methods: To describe intra-DTU diversity, we developed a highly resolutive maxicircle multilocus sequence typing (mtMLST) scheme, which was evaluated in comparison with current nuclear typing tools using isolates belonging to the oldest and most widely occurring lineage TcI. In parallel, we exploited read depth data, generated by Illumina sequencing of the maxicircle genome from the TcI reference strain to investigate the existence of mitochondrial heteroplasmy (heterogeneous mitochondrial genomes in an individual cell) in T. cruzi and resolve its potential role as a source of genotyping error. Results: Comparison of nuclear and mitochondrial data uncovered multiple novel mitochondrial introgression events among different geographical populations from Bolivia, Venezuela and Colombia as well as between majorT. cruzi DTUs. Illumina sequencing data from the TcI genome strain revealed multiple different mitochondrial genomes within an individual parasite (heteroplasmy) that were, however, not sufficiently divergent to represent a major source of typing error. Conclusion: mtMLST provides a powerful approach to genotyping at the sub-DTU level. This strategy will facilitate attempts to resolve phenotypic variation in T. cruzi and to address epidemiologically important hypotheses in conjunction with intensive spatio-temporal sampling. The observations of both general and specific incidences of nuclear-mitochondrial phylogenetic incongruence indicate that genetic recombination is geographically widespread and continues to influence the natural population structure of TcI, a conclusion which challenges the traditional paradigm of clonality in T. cruzi.