Trypanosoma cruzi (Tc), the causative agent of Chagas disease, is a diverse species with 2 primary genotypes, TcI and TcII, with TcII further subdivided into 5 subtypes (IIa-e). This study evaluated infection dynamics of 4 genetically and geographically diverse T. cruzi strains in 2 South American reservoirs, degus (Octodon degus) and grey short-tailed opossums (Monodelphis domestica). Based on prior suggestions of a genotype-host association, we hypothesized that degus (placental) would more readily become infected with TcII strains while short-tailed opossums (marsupial) would be a more competent reservoir for a TcI strain. Individuals (n=3) of each species were intraperitoneally inoculated with T. cruzi trypomastigotes of TcIIa [North America (NA)-raccoon (Procyon lotor) origin], TcI [NA-Virginia opossum (Didelphis virginiana)], TcIIb [South America (SA)-human], TcIIe (SA-Triatoma infestans), or both TcI and TcIIa. Parasitaemias in experimentally infected degus peaked earlier (7-14 days post-inoculation (p.i.)) compared with short-tailed opossums (21-84 days p.i.). Additionally, peak parasitaemias were higher in degus; however, the duration of detectable parasitaemias for all strains, except TcIIa, was greater in short-tailed opossums. Infections established in both host species with all genotypes, except for TcIIa, which did not establish a detectable infection in short-tailed opossums. These results indicate that both South American reservoirs support infections with these isolates from North and South America; however, infection dynamics differed with host and parasite strain.