The pattern of flavivirus infection in mosquitoes belonging to the genus Aedes and Culex collected in two regions of north-eastern Italy (Trentino and Veneto) was assessed. Mosquitoes were collected during 2012 and screened for Flavivirus using a generic RT-nested-PCR targeted on a region of the nonstructural 5 (NS5) gene. The phylogenetic analysis was performed on a fragment of around 1000 bp. Virus isolation was attempted in C6/36 insect cell lines and the infected cell cultures were studied by electron microscopy. We detected a wide distribution of Aedes Flavivirus (AeFV) in Aedes (Ae.) albopictus, with higher infection prevalence in Trentino than in Veneto. In Culex (Cx.) pipiens collected in Veneto, we detected Usutu virus (USUV) and a new sequence of an insect-specific flavivirus. Interestingly, we detected for the first time AeFV in Cx. pipiens, in both regions. Viral isolation in cell culture was successful for AeFV. AeFV sequences found in Veneto showed a high percentage of similarity to those detected in Trentino and to those previously reported in other areas of northern Italy. Coinfections with different flaviviruses were not detected.