Abstract Background During recent years, numerous novel ‘insect flaviviruses’ have been discovered in natural mosquito populations. In a previous study we described the presence of flavivirus DNA sequences integrated in Aedes albopictus (Asian tiger mosquito) populations from Northern Italy in 2007. Methods During 2008 we collected and tested Aedes females for flavivirus presence and developed phylogenetic analysis, virus isolation, electron microscopy studies and RNAse treatments. Results We detected a high prevalence of flavivirus in Ae. albopictus (77.5%). The phylogenetic analysis identified the insect flavivirus sequences as Aedes flavivirus (AEFV) recently described in Japan, and that may have been introduced in Italy travelling with the tiger mosquito. Some of these pools grew in C6/36 cells, producing cytopathic effects, and the RNase treatment results showed the presence of the detected sequences in RNA forms. Furthermore, we detected a new insect flavivirus in one pool of Aedes cinereus/geminus mosquitoes. Phylogenetic analysis of this virus shows that it forms a distinct cluster within the clade of insect flavivirus. Conclusions This is the first study to report a high prevalence, to describe the seasonal activity and an isolation of the insect flavivirus Aedes flavivirus in Europe. Moreover we describe the detection of a new insect flavivirus detected from Ae. cinereus mosquitoes from Italy. These flavivirus may be common, ubiquitous and diverse in nature and we discuss the implications of the insect flavivirus group in virus evolution and transmission.