A four-year longitudinal study of the structure of sylvatic transmission cycles of Trypanosoma cruzi, reservoir host competence and parasite discrete typing units was conducted in a disturbed rural area of the humid Chaco in Argentina. Among 190 mammals examined by xenodiagnosis and polymerase chain reaction amplification, the composite prevalence of infection was substantially higher in Dasypus novemcinctus armadillos (57.7%) and Didelphis albiventris opossums (38.1%) than in Euphractus sexcinctus (20.0%), Tolypeutes matacus (12.5%), and Chaetophractus vellerosus (6.3%) armadillos. Trypanosoma cruzi was detected for the first time in Thylamys pusilla small opossums and in two unidentified small rodents. Infection was spatially aggregated only in armadillos. All Didelphis were infected with T. cruzi I and all armadillo species were infected with T. cruzi III, implying two distinct sylvatic cycles with no inputs from the domestic cycle. Dasypus armadillos and Didelphis opossums were much more infectious to vectors than other armadillos, small opossums, or rodents.