AbstractVertical transmission in Aedes aegypti and Aedes albopictus is considered a maintenance mechanism for dengue virus (DENV) during unfavorable conditions and may be implicated in dengue outbreaks. Since DENV infection dynamics vary among wild-type viruses and vector populations, vertical transmission rates can also vary between regions. However, even though São Paulo is the most populous city in the Americas and has experienced major dengue epidemics, natural vertical transmission had never been detected in this area before. Here we confirm and describe for the first time natural vertical transmission of DENV-3 in two pools of male Ae. albopictus from the city of São Paulo. The detection of DENV-3 in years when no human autochthonous cases of this serotype were recorded suggests that silent circulation of DENV-3 is occurring and indicates that green areas may be maintaining serotypes that are not circulating in the human population, possibly by a vertical transmission mechanism.