Aedes aegypti (L.) is the principal mosquito vector of dengue fever, the second-most deadly vector-borne disease in the world. In Ae. aegypti and other arthropod disease vectors, genetic markers can be used to inform us about processes relevant to disease spread, such as movement of the vectors across space and the temporal stability of vector populations. In late 2009, 27 locally acquired cases of dengue fever were reported in Key West, FL. The last dengue outbreak in the region occurred in 1934. In this study, we used 12 microsatellite loci to examine the genetic structure of 10 Ae. aegypti populations from throughout the Florida Keys and Miami to assess gene flow along the region's main roadway, the Overseas Highway. We also assessed temporal genetic stability of populations in Key West to determine whether the recent outbreak could have been the result of a new introduction of mosquitoes. Though a small amount of geographic genetic structure was detected, our results showed high overall genetic similarity among Ae. aegypti populations sampled in southeastern Florida. No temporal genetic signal was detected in Key West populations collected before and after the outbreak. Consequently, there is potential for dengue transmission across southeastern Florida; renewed mosquito control and surveillance measures should be taken.