We recently discovered several insect-specific flaviviruses (ISFs) in mosquitoes from different regions of Australia. These viruses do not replicate in vertebrate cells but grow to high titre in mosquito cultures. Genome sequence analyses of these viruses revealed we had discovered 7 new ISF species representing two distinct genetic lineages. Construction of infectious DNAs and chimeric viruses allowed us to identify stages at pre- and post-cell entry where ISF infection and replication is blocked in vertebrate cells. We also generated a series of chimeric viruses expressing the structural genes (prM-E) from pathogenic flaviviruses, including West Nile, Zika and dengue viruses, spliced into the genetic backbone of two different ISF species. These chimeras also exhibited the insect-specific phenotype of their parental ISFs, growing efficiently in mosquito cells but not in vertebrate cultures. However, EM and extensive antigenic analysis of the chimeric virions revealed they were structurally indistinguishable from virions of the pathogenic parental viruses that donated the structural genes. These chimeric viruses thus provide potential candidates as safe diagnostic antigens or vaccines for mosquito-borne flaviviral diseases.