Chagas disease, a significant public health concern in the Americas, is caused by a protozoan parasite, Trypanosoma cruzi. The life cycle of T. cruzi involves kissing bugs (Triatoma spp.) functioning as vectors and mammalian species serving as hosts. Raccoons (Procyon lotor) and opossums (Didelphis virginiana) have been identified as important reservoir species in the life cycle of T. cruzi, but prevalence in both species in the southeastern US is currently understudied. We quantified T. cruzi prevalence in these two key reservoir species across our study area in South Carolina, US, and identified factors that may influence parasite detection. We collected whole blood from 183 raccoons and 126 opossums and used PCR to detect the presence of T. cruzi. We then used generalized linear models with parasite detection status as a binary response variable and predictor variables of land cover, distance to water, sex, season, and species. Our analysis indicated that raccoons experienced significantly higher parasite detection rates than Virginia opossums, with T. cruzi prevalence found to be 26.5% (95% confidence interval [CI], 20.0–33.8) in raccoons and 10.5% (95% CI, 5.51–17.5) in opossums. Overall, our results concur with previous studies, in that T. cruzi is established in reservoir host populations in natural areas of the southeastern US.