Phlebotomine sand flies are hematophagous insect vectors of the protozoan parasites of the genus Leishmania Ross, 1903 (Kinetoplastida: Trypanosomatida) that infect mammals, including humans, causing leishmaniasis. In Porto Alegre, Brazil, three autochthonous cases of human visceral leishmaniasis were reported in 2016 through 2017. We analyzed for the presence of Leishmania DNA in sand flies collected at the neighborhood of Agronomia, Porto Alegre, Brazil. Phlebotomine sand flies were collected at three sites from October 2014 to September 2015. Female sand flies were pooled in numbers from 1 to 20 depending upon species, locality, and date; all were screened for Leishmania infection by the amplification of the ITS1 region. In total 518 phlebotomine sand flies were collected: Psathyromyia lanei (Barretto and Coutinho, 1941) (Diptera: Psychodidae) (30.5%), Brumptomyia sp. (França and Parrrot, 1921) (Diptera: Psychodidae) (25.7%), Migonemyia migonei (Franca, 1920) (Diptera: Psychodidae) (21.4%), Pintomyia fischeri (Pinto, 1926) (Diptera: Psychodidae) (21.4%), and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae) (1.0%). Most sand flies were collected during the hot and rainy season from October 2014 to April 2015. Of the 113 pools analyzed, five pools of Pi. fischeri were PCR-positive with the amplicons possessing sequences similar (>95%) to that of Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatida). These results represent the first molecular detection of Le. infantum in Pi. fischeri. It is possible that Pi. fischeri is involved in the transmission cycle of Le. infantum in the studied area; however, further studies are needed to establish the true role of Pi. fischeri in the visceral leismaniasis cycle.