Most flaviviruses reported to date are classified as arboviruses based on their replication in both vertebrate and arthropod hosts. A novel group of insect-specific flaviviruses (ISFs), that replicate only in mosquitoes and appear to spread through insect populations via vertical transmission, have recently been detected in a range of mosquito species from around the world. Recently we discovered the first Australian ISF (Palm Creek virus – PCV) in populations of Coquillettidia xanthogaster mosquitoes from Darwin. Genetic and antigenic analysis of this virus revealed that PCV represented a new species of ISF and clustered with other members of this group isolated from North America, Asia and Africa. Further screening of mosquito samples from North-Western Australia, The Gulf of Carpentaria and Sydney have revealed the presence of additional novel ISFs from Aedes vigilax and Anopheles merakensis mosquitoes that were genetically distinct from PCV and all other known ISFs. Our demonstration that prior infection with PCV rendered mosquito cells less competent to replicate the pathogenic flaviviruses Murray Valley encephalitis virus and West Nile virus, suggests that replication of the latter viruses was regulated by a superinfection exclusion mechanism. In light of these results, it is tempting to speculate that this new group of viruses may influence the ability of some vectors to transmit flaviviruses of medical and veterinary significance and provide an interesting opportunity to study the mechanism of their maintenance and spread.