The continuing worldwide increase in urbanization can potentially have a major impact on the epidemiology of vector-borne diseases, as anthropogenic changes to the environment are known to favor a few species of mosquitoes that can thrive in urban environments. Culex quinquefasciatus Say (Diptera: Culicidae) is found extensively in urban habitats, where it lives in degraded, polluted areas and is often the only species of mosquito capable to surviving under such conditions. Traditional mosquito control strategies no longer have the desired effect due to the several factors such as insecticide resistance, abundance of breeding sites, lack of proper sewage and sanitation, and absence of natural predator, leading Cx. quinquefasciatus populations to increase its numbers in cities. In this study, five Cx. quinquefasciatus populations were analyzed using 12 microsatellite markers to investigate whether the dynamics of these populations are being modulated by urbanization and how they are structured in the city of São Paulo, Brazil. Our results indicate that only one of the study populations (the population from Anhanguera Park) exhibited evidence of expansion.The populations from Ibirapuera Park and Piqueri Park, the most urbanized regions of the areas studied, did not show signs of expansion. A better understanding of the mechanisms involved in the dispersal of Cx. quinquefasciatus and its colonization of new areas, as well as the species' demographic patterns and how these are associated with urbanization, particularly in areas undergoing a rural-to-urban transformation, such as Anhanguera Park, is of great importance for mosquito control.