Background: Trypanosoma cruzi (Kinetoplastida, Trypanosomatidae) infection is an ancient and widespread zoonosis distributed throughout the Americas. Ecologically, Brazil comprises several distinct biomes: Amazonia, Cerrado, Caatinga, Pantanal and the Atlantic Forest. Sylvatic T. cruzi transmission is known to occur throughout these biomes, with multiple hosts and vectors involved. Parasite species-level genetic diversity can be a useful marker for ecosystem health. Our aims were to: investigate sylvatic T. cruzi genetic diversity across different biomes, detect instances of genetic exchange, and explore the possible impact of ecological disturbance on parasite diversity at an intra-species level. Methods: We characterised 107 isolates of T. cruzi I (TcI; discrete typing unit, DTU I) from different major Brazilian biomes with twenty-seven nuclear microsatellite loci. A representative subset of biologically cloned isolates was further characterised using ten mitochondrial gene loci. We compared these data generated from Brazilian TcI isolates from around America. Results: Genetic diversity was remarkably high, including one divergent cluster that branched outside the known genetic diversity of TcI in the Americas. We detected evidence for mitochondrial introgression and genetic exchange between the eastern Amazon and Caatinga. Finally, we found strong signatures of admixture among isolates from the Atlantic Forest region by comparison to parasites from other study sites. Conclusions: Atlantic Forest sylvatic TcI populations are highly fragmented and admixed by comparison to others around Brazil. We speculate on: the possible causes of Atlantic Forest admixture; the role of T. cruzi as a sentinel for ecosystem health, and the impact disrupted sylvatic transmission cycles might have on accurate source attribution in oral outbreaks.