To identify naturally infected Lutzomyia spp. by Leishmania (Viannia) braziliensis, a PCR multiplex non-isotopic hybridisation assay was developed for the analysis of insect samples collected in distinct areas of the municipality of Rio de Janeiro (Brazil), from March to December 2003. Data from experimental infection indicate that the method can detect one individual infected insect out of ten. Wild sand flies were classified and grouped into pools of 10 specimens each, reaching a total of 40 female groups. Positive results were obtained with pools of Lu. intermedia (5/32) and Lu. migonei (3/5) collected in two areas from the district of Jacarepaguá presenting recent cases of human and canine leishmaniasis. Considering eight infected groups (8/40) with at least one positive insect in each, it was possible to infer an infection rate of 2%. This technique permits the synchronous processing of a large number of samples, in order to investigate infection rates in sand fly populations and to identify potential insect vectors. The results presented here represent the first molecular approach used to infer the natural infection index in both Lutzomyia spp. and constitute essential data to the understanding of leishmaniasis ecoepidemiology in endemic areas from Rio de Janeiro.