Chagas disease or American trypanosomiasis is an infection caused by the parasite Trypanosoma cruzi. According to its genetic characteristics, this parasite is divided into six groups (TcI-TcVI) called discrete typing units (DTUs). Trypanosoma cruzi is transmitted to humans by insects of the Triatominae family. In Mexico, despite having a great variety of triatomine species, little is known about vector sylvatic populations and the DTUs associated with them. In this work, molecular markers such as minicircle, miniexon, 18S, and 24S ribosomal genes and restriction fragment length polymorphism (RFLP) analysis of the hsp70 gene were used to determine the DTUs present in vectors from rural communities and sylvatic areas inside the Biosphere Reserve Los Tuxtlas, Veracruz, in southeast Mexico. One hundred triatomines were collected and two species were identified: Triatoma dimidiata and Panstrongylus rufotuberculatus. The infection with T. cruzi was determined in 29% of analyzed vectors from the domestic area and TcI was the predominant DTU. Furthermore, 71% of vectors from the sylvatic environment were infected and TcI, TcII, TcIV, and TcVI were identified. One female and one male of P. rufotuberculatus were infected only with TcI. This is the first report of TcVI in T. dimidiata from the sylvatic area in México and the first report of P. rufotuberculatus infected with T. cruzi in Mexico.