In this study, we sought to identify sand fly vectors of the Leishmania species that circulate in distinct eco-epidemiological disease-endemic rural areas within the Espírito Santo State in southeastern Brazil. PCR amplification of a conserved region of the minicircle kDNA was used to estimate infection rates in field-captured, peridomestic female sand flies. Only 13 of the 1689 female sand fly specimens (0.77%) actually contained Leishmania DNA. Leishmania braziliensis infections were found in Lutzomyia intermedia and Lu. whitmani, and, for the first time, in Lu. fischeri and Lu. ferreirana. Interestingly, the high rate of genetic polymorphism of the L. braziliensis parasites in one of the disease-endemic areas that were studied may reflect specific transmission cycles involving different sand fly vectors.