Studies on natural infection by Leishmaniaspp of sandflies collected in endemic and nonendemic areas can provide important information on the distribution and intensity of the transmission of these parasites. This study sought to investigate the natural infection by Leishmaniain wild female sandflies. The specimens were caught in the city of Corumbá, state of Mato Grosso do Sul (Brazil) between October 2012-March 2014, and dissected to investigate flagellates and/or submitted to molecular analysis to detect LeishmaniaDNA. A total of 1,164 females (77.56% of which were Lutzomyia cruzi) representing 11 species were investigated using molecular analysis; 126 specimens of Lu. cruziwere dissected and also submitted to molecular analysis. The infection rate based on the presence of LeishmaniaDNA considering all the sandfly species analysed was 0.69%; only Leishmania (Leishmania) amazonensiswas identified in Lu. cruziby the molecular analysis. The dissections were negative for flagellates. This is the first record of the presence of L. (L.) amazonensisDNA in Lu. cruzi, and the first record of this parasite in this area. These findings point to the need for further investigation into the possible role of this sandfly as vector of this parasite.