Randomly amplified polymorphic DNA (RAPD) analysis was performed to infer the magnitude and pattern of genetic differentiation among Lutzomyia (Nyssomyia) intermedia populations from two ecologic regions in southeast Brazil where transmission of American cutaneous leishmaniasis (ACL) by L. (Viannia) braziliensis reflects distinct eco-epidemiologic features. Two hundred thirty-five bands from six primers were analyzed for genetic variation within and between sand fly populations. A lower level of intrapopulational variability was found in domestic sand flies (compared with the peridomestic population). Gene flow FST estimates indicated homogeneity in the studied population, with limited population substructuring, but with a tendency of sand fly vectors to form clusters by micro-habitat (domestic and peridomestic). The level of genetic structuring of Lu. intermedia population from each ecologic region is distinct and may reflect association or independence between the domestic and the peridomestic habitats in rural and periurban areas, respectively, both reflecting distinct characteristics of the transmission cycle of ACL.