Chagas disease, also known as American trypanosomiasis, is caused by the parasite Trypanosoma cruzi and is transmitted in the feces of insect-vector species in the taxonomic sub-family Triatominae. As a neglected tropical disease, the disease is endemic to the tropical Americas, has no cure, and disproportionately afflicts rural and impoverished communities throughout Latin America. The objective of our study was to evaluate biological factors contributing to the spread of Chagas disease in eastern El Salvador by using PCR techniques to identify blood-feeding preferences of Triatoma dimidiata inside and outside of housing environments. We hypothesized that T. dimidiata blood-feeding preferences would differ with respect to habitat (domiciliary or peridomiciliary), development stage (instars or adults), T. dimidiata infection-status with the T. cruzi parasite (positive or negative), and the sex of T. dimidiata. We found that T. dimidiata in peridomiciliary environments preferred feeding on birds, dogs, and rats, but were more likely to be at the adult-stage when positive for T. cruzi infection. In contrast, T. dimidiata in domiciliary environments preferred feeding on pigs, humans, and mice, but were more likely to be at instar-stages when positive for T. cruzi infection. Additionally, female T. dimidiata preferred feeding on birds and dogs, while males preferred feeding on pigs, humans, and rats. We discuss differences between our findings and those reported from western El Salvador and highlight the importance of understanding the vector ecology of T. dimidiata as part of a broader EcoHealth strategy to mitigate the transmission of Chagas disease.