Background: Triatomines in Latin America are natural Chagas disease (ChD) vectors. Triatomine domiciliation is one of the main factors increasing the occurrence risk of this disease in humans. There are 66 triatomine species in Brazil, with three genera of significant epidemiological importance—Panstrongylus, Rhodnius, and Triatoma. Among the Triatoma species, Triatoma williami, a wild species, has been reported in Goiás, Mato Grosso, and Mato Grosso do Sul. In the Barra do Garças, Mato Grosso, the invasion by triatomines has been reported, with T. williami being the most common species. This study aimed to survey triatomine fauna and determine the Trypanosoma cruzi natural infection rates in triatomines in the urban area of Barra do Garças, Mato Grosso, Brazil. Methods: Triatomine specimens were sampled by passive surveillance or active search by agents combating endemic diseases from 2019 to 2020. A parasitological feces diagnosis was performed to detect the presence of T. cruzi after the specimens were identified. Concerning T. cruzi identification, molecular diagnosis and genetic sequencing were performed to determine the strain, also called discrete typing units (DTUs). Results: The 211 triatomines were collected, distributed in specimens of T. williami (84.4%), P. geniculatus (3.3%), P. diasi (1.4%), and R. neglectus (10.9%). Two colonies of T. williami were found through morphological analyses. These insects were sampled inside domiciles in an urban area neighboring Jardim Pitaluga (15° 51′57.7″ N, 052° 16′ 04.5 E). The records were sampled in September 2019 and January 2021. The rate of natural infection by T. cruzi was 39.4%. Two T. williami specimens from the sampled colonies were positive for the T. cruzi strain DTU IV. Conclusions: This is the first time that T. williami has been confirmed in an urban area of Barra do Garças, Mato Grosso, Brazil. Further studies are needed for a clearer understanding of the ecology of this species for prevention and control mechanisms since its sampled specimens had a high rate of natural infection by T. cruzi.