Flaviviruses are responsible for considerable disease in humans and animals globally. While these viruses generally cycle between arthropod vectors and vertebrate hosts, studies on insect-specific flaviviruses (ISFs) suggest this subset of flaviviruses are transmitted only between mosquitoes. Recently, our group isolated Palm Creek virus (PCV) from Darwin and determined it to be the prototype Australian ISF. In vitro analysis of PCV revealed it can suppress the replication of the human pathogens Murray Valley encephalitis (MVE) and West Nile virus (WNV) in co-infected mosquito cells. This finding emphasizes the importance of further characterising the diversity of ISFs within Australia. We subsequently assessed additional mosquito populations from New South Wales for the presence of ISFs and other insect-specific viruses. One hundred homogenized mosquito pools collected from urban areas of Sydney were inoculated onto C6/36 cells and screened by ELISA using a cocktail of novel monoclonal antibodies reactive to antigens associated with infection by ISFs and a range of other mosquito-borne viruses. ELISA positive samples were then subjected to a pan-flavivirus RT-PCR. Thirty-eight isolates of a novel ISF from samples of Aedes vigilax mosquitoes were obtained. Viral genome sequencing and phylogenetic analysis revealed that we had discovered a new species of flavivirus and the first ISF to be isolated from Ae. vigilax mosquitoes. We have tentatively named the virus ‘Silverwater virus’ (SwV) based on the location of its isolation in Sydney. In vitro and in vivo phenotypic characterisation of the virus is in progress.