In this study, we investigated the presence of Leishmania in sand flies collected from a peridomestic area in Corumbá, Mato Grosso do Sul, after an autochthonous case of cutaneous leishmaniasis was confirmed. A total of 1,542 sand flies belonging to seven species were collected, with Lu. cruzi being the most prevalent (94.3%). We detected the presence of DNA from Le. infantum (7 pools) and Le. braziliensis (3 pools) by sequencing the ITS1 amplicon in ten pools, all of which were composed of engorged (3) and non-engorged (7) females of Lu. cruzi. We collected 24 engorged females, with Homo sapiens being the most common blood meal source (91.6%), followed by Dasyprocta azarae and Canis lupus familiaris (4.2% each). To our knowledge, this is the first molecular evidence of Le. braziliensis in wild-caught Lu. cruzi in Brazil, suggesting its potential role as a vector for this parasite.