Aedes aegypti (L.) (Diptera: Culicidae), the main vector of yellow fever and dengue viruses, was eradicated from Argentina between 1955 and 1963, but reinvaded the country in 1986. In Uruguay, the species was reintroduced in 1997. In this study we used highly polymorphic inter-simple sequence repeats (ISSR) markers to analyse the genetic structure of Ae. aegypti populations from Uruguay and northeastern Argentina to identify possible colonization patterns of the vector. Overall genetic differentiation among populations was high (F(ST) = 0.106) and showed no correlation with geographic distance, which is consistent with the short time since the reintroduction of the species in the area. Differentiation between pairs of Argentine populations (F(ST) 0.072 to 0.221) was on average higher than between Uruguayan populations (F(ST)-0.044 to 0.116). Bayesian estimation of population structure defined four genetic clusters and most populations were admixtures of two of them: Mercedes and Treinta y Tres (Uruguay) were mixtures of clusters 1 and 3; Salto (Uruguay) and Paraná (Argentina) of clusters 1 and 4; Fray Bentos (Uruguay) of clusters 2 and 3, and Gualeguaychú (Argentina) of clusters 2 and 3. Posadas and Buenos Aires in Argentina were fairly genetically homogeneous. Our results suggest that Ae. aegypti recolonized Uruguay from bordering cities in Argentina via bridges over the Uruguay River and also from Brazil.