We characterized 28 new isolates of Trypanosoma cruzi IIc (TCIIc) of mammals and triatomines from Northern to Southern Brazil, confirming the widespread distribution of this lineage. Phylogenetic analyses using cytochrome b and SSU rDNA sequences clearly separated TCIIc from TCIIa according to terrestrial and arboreal ecotopes of their preferential mammalian hosts and vectors. TCIIc was more closely related to TCIId/e, followed by TCIIa, and separated by large distances from TCIIb and TCI. Despite being indistinguishable by traditional genotyping and generally being assigned to Z3, we provide evidence that TCIIa from South America and TCIIa from North America correspond to independent lineages that circulate in distinct hosts and ecological niches. Armadillos, terrestrial didelphids and rodents, and domestic dogs were found infected by TCIIc in Brazil. We believe that, in Brazil, this is the first description of TCIIc from rodents and domestic dogs. Terrestrial triatomines of genera Panstrongylus and Triatoma were confirmed as vectors of TCIIc. Together, habitat, mammalian host and vector association corroborated the link between TCIIc and terrestrial transmission cycles/ecological niches. Analysis of ITS1 rDNA sequences disclosed clusters of TCIIc isolates in accordance with their geographic origin, independent of their host species.