We present a population genetic study of Aedes aegypti in Brazil using isoenzyme markers. Four polymorphic loci were used to examine 11 mosquito collections at four periods in 2003. Samples from a dengue-endemic area (southeastern region) and a dengue-free area (southern region) connected by an important network of roads and railways were analyzed. The degree of genetic differentiation observed between populations is consistent with limited gene flow between them. There was no evidence of passive dispersion of Ae. aegypti by vehicles among the different routes linking metropolitan areas.