Simple SummaryThe mosquitoAedes(Stegomyia)aegyptirepresents a major challenge for public health due to its proliferation in many regions of the world and its capacity to disperse arboviruses. The verification of the genetic characteristics of this mosquito and the differentiation of these characteristics both within and between its populations in the Mid-North region of Brazil will be fundamentally important for the development of effective measures for the control of this disease vector. Previous studies of mitochondrial markers in the Mid-North region revealed two genetic lineages ofAe. aegypti, and high levels of genetic differentiation, which represent a potential risk for the occurrence of epidemics and a major problem for local public health authorities. Using microsatellite markers, the present study identified distinct genetic lineages ofAe. aegyptiin areas with high rates of infestation. This analysis revealed the coexistence of two different genetic lineages, which were highly differentiated in genetic terms. These findings on the genetic status of the populations will be extremely important for the development of innovative strategies for the control of the populations of this disease vector, and effective measures for the protection of local human populations.AbstractAedes aegyptiis the principal vector of the arboviruses—yellow fever, dengue virus, chikungunya, and zika virus. Given the epidemiological importance of this mosquito, its capacity to adapt to different habitats, and its resistance to many types of control measures, systematic research into the genetic variability of the populations of this mosquito is one of the most important steps toward a better understanding of its population structure and vector competence. In this context, the present study verified the presence of distinct genetic lineages ofAe. aegyptiin areas with high infestation rates, based on the analysis of microsatellite markers. The samples were collected in nine municipalities with high building infestation rates in the Mid-North region of Brazil. Six microsatellite loci were genotyped in the 138 samples, producing a total of 32 alleles, varying from one to nine alleles per locus in each of the different populations. The AMOVA revealed greater within-population genetic differentiation with high fixation rates. The general analysis of population structure, based on a Bayesian approach, revealed K = 2, with twoAe. aegyptilineages that were highly differentiated genetically. These data on the connectivity of the populations and the genetic isolation of the lineages provide important insights for the development of innovative strategies for the control of the populations of this important disease vector.