To identify Lutzomyia ( Nyssomyia ) neivai naturally infected by Leishmania a multiplex polymerase chain reaction (PCR) was used for the analysis of 450 specimens (270 females, 180 males) collected in an endemic periurban area of cutaneous leishmaniasis in Porto Alegre, Brazil. Insects were grouped into pools of 10 and positive results were achieved in 3/27 Lu . ( N .) neivai female pools. Infection by L . ( Viannia ) braziliensis was confirmed after hybridizing PCR products with a subgenus–specific biotinylated probe. Considering the detection of three positive pools with at least one infected insect in each, an infection rate of 1.1% was estimated. Our results associated with epidemiologic data suggest a potential ability of Lu . ( N .) neivai in transmitting L . braziliensis in Porto Alegre, where the first notifications of autochthonous cutaneous leishmaniasis in humans occurred in 2002, with an increase in the number of cases in recent years possibly as a consequence of deforestation and agricultural activities in the area.