Flaviviruses such as West Nile virus (WNV), dengue virus and Zika virus are responsible for significant mosquito-borne disease. However recently, insect-specific flaviviruses (ISF) which only replicate in mosquito hosts have been identified. Of particular interest, mosquitoes persistently infected with some of these ISFs are less capable of transmitting pathogenic flaviviruses. This study aimed to generate infectious cDNA constructs to identify the genes responsible for host restriction in ISFs. Circular polymerase extension cloning (CPEC) was used to generate infectious DNA constructs using a modified insect (OpIE2) promoter to drive transcription in C6/36 insect cells. These infectious DNA constructs were transfected into cells and characterised using a combination of IFA, RT-PCR and ELISA. Viral authenticity was confirmed using sequencing and antibodies specific to each of the viruses. Infectious virus was successfully recovered from C6/36 mosquito cells transfected with a cDNA constructs of Australian ISFs, Parramatta river virus (PaRV) and Palm Creek virus (PCV). A similar infectious cDNA under transcriptional control of a mammalian promoter was used to directly transfect mouse embryo fibroblasts and revealed that, in both the absence or presence of a type I IFN response, PaRV did not initiate replication in vertebrate cells even when cell attachment and virion entry steps were by-passed. We also report the generation and characterization of a viable chimeric virus containing PCV structural proteins on a WNV backbone, and the first reported chimeric flaviviruses with WNV, DENV and ZIKV structural proteins on a PCV backbone. These infectious ISF DNA constructs provide novel tools to elucidate the mechanisms restricting ISFs to mosquito hosts and are currently being manipulated to identify the viral host factors associated with this restriction. These studies will also deliver novel insights into flavivirus evolution.