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Background: Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi and vectored by triatomines, affects millions of people worldwide. In endemic countries including Mexico, infections in domestic animals, such as dogs, may affect the risk of human disease when they serve as a source of infection to vectors that subsequently infect humans. Materials and Methods: We conducted a cross-sectional study of 296 dogs from two cities near the northern and southern borders of Mexico: Reynosa, Tamaulipas, and Tuxtla Gutierrez, Chiapas. Infection was measured based on testing of blood using T. cruzi quantitative PCR (qPCR) and up to three antibody detection assays. The StatPak immunochromatographic assay was used to screen samples and the indirect fluorescent antibody (IFA) and multiplex microsphere immunoassay (MIA) tests were used as secondary tests on all samples that screened positive and a subset of negatives. Serologic positivity was defined based on reactivity on at least two independent tests. Results: Of the 280 samples tested for parasite DNA, two (0.7%) were positive, one of which (0.4%) was confirmed as T. cruzi discrete typing unit TcIV. Overall, 72 (24.3%) samples were reactive for T. cruzi antibodies via StatPak of which 8 were also positive using MIA and 2 were also positive using IFA (including one of the PCR-positive dogs). Overall, nine dogs (3.4%) met study criteria of positivity based on either/both serology or PCR tests. Positive dogs were found in both regions of Mexico; five (2.7%) from Reynosa and four (3.6%) from Tuxtla Gutierrez. We found no association between infection status and state of origin, sex, age group, breed group, neighborhood, and whether other pets lived in the home. Conclusion: Our results re-emphasize dogs' utility as sentinels for T. cruzi in Mexico and underscore the need for improved veterinary diagnostic tests and parasite surveillance at the household level in endemic countries.