The purpose of this study was to conduct an entomological analysis, determination of feeding sources, and molecular identification of triatomines in five communities of the Istmo de Tehuantepec, Oaxaca. The only found species in two of five searched communities (San Mateo del Mar and Tehuantepec City) was Triatoma phyllosoma. Colonization indices were high in both communities. In San Mateo del Mar, the insects were found indoors and in Tehuantepec City in peridomestic areas. The Trypanosoma cruzi infection indices were 2.1% in San Mateo del Mar and 39.4% in Tehuantepec City. This difference could be related to the high numbers of triatomine feeding on hens in the former community. In contrast, in Tehuantepec, dogs were the principal triatomine feeding sources. All nymphs and adults that were genetically analyzed belonged to the species T. phyllosoma. Low levels of genetic variation were found between vectors from both communities.