Aedes aegypti L. (Diptera, Culicidae) is the main vector of dengue and yellow fever. In Argentina, the species was apparently eradicated approximately in 1964; by 1986, it was reintroduced. To identify different gene pools in geographical populations of the species and to ascertain the possible routes of colonization, we analyzed the diversity of mitochondrial DNA haplotypes in 572 specimens from Argentina and neighboring countries. We found that the restriction fragment length polymorphism-polymerase chain reaction screening of a large DNA fragment including the A T-rich region was the best strategy to reconstruct the colonization pattern of Ae. aegypti in Argentina. Twenty haplotypes were recognized; levels of genetic similarity varied among populations from different geographical locations. The haplotype network constructed on the basis of genetic distances showed three well differentiated groups. Two of them exhibited a well defined spatial distribution and populations in these groups presented an isolation-by-distance pattern. The persistence of relictual populations after the last eradication campaigns would explain the high levels of haplotype diversity and the presence of exclusive haplotypes in urban centers from northwestern Argentina. Eastern Argentine populations showed one prevalent haplotype, also predominant in Brazil and Paraguay. Our results highlight the need for efficient surveys and control campaigns, given the strong effect of land trade on genetic exchange among mosquito populations from Argentina and neighboring countries where dengue is endemic.