© 2015. To date, insect-specific flaviviruses (ISFs) have only been isolated from mosquitoes and increasing evidence suggests that ISFs may affect the transmission of pathogenic flaviviruses. To investigate the diversity and prevalence of ISFs in Australian mosquitoes, samples from various regions were screened for flaviviruses by ELISA and RT-PCR. Thirty-eight pools of Aedes vigilax from Sydney in 2007 yielded isolates of a novel flavivirus, named Parramatta River virus (PaRV). Sequencing of the viral RNA genome revealed it was closely related to Hanko virus with 62.3% nucleotide identity over the open reading frame. PaRV failed to grow in vertebrate cells, with only Aedes-derived mosquito cell lines permissive to replication, suggesting a narrow host range. 2014 collections revealed that PaRV had persisted in A. vigilax populations in Sydney, with 88% of pools positive. Further investigations into its mode of transmission and potential to influence vector competence of A. vigilax for pathogenic viruses are warranted.