Culex annulirostris Skuse, Culex palpalis Taylor and Culex sitiens Wiedemann are members of the Culex sitiens subgroup that exist in Australasia. They are widely distributed and regularly comprise over the half of mosquitoes collected from arbovirus surveys and field collections in Australia and PNG. From these mosquitoes, Cx. annulirostris is the major vector of endemic arboviruses in Australia and is also responsible for the establishment of the Japanese encephalitis virus (JEV) in southern Papua New Guinea (PNG) as well as JEV incursions into northern Australia. Papua New Guinea and mainland Australia are separated by a small stretch of water, the Torres Strait, and its islands. While there has been regular JEV activity on these islands, JEV has not established on mainland Australia despite an abundance of its vector Cx. annulirostris and porcine amplifying hosts. Despite the public health significance of this mosquito and the fact that its adults show overlapping morphology with close relative Cx. palpalis, its evolution and genetic structure remain undetermined. I address a hypothesis that there is significant genetic diversity in Cx. annulirostris and that the identification of this diversity will shed light on the paradox that JEV can cycle on an island 70 km from mainland Australia while not establishing in Australia itself. My study assessed the biodiversity within Cx. annulirostris and Cx. palpalis using a extensive collection of mosquitoes and analysing both mitochondrial (COl) and a nuclear (ace-2) markers using phylogenetic reconstruction and hypothesis testing. I provide evidence of subdivision of these two species into several geographically delimited lineages, which indicates the presence of highly divergent populations or cryptic species. However I found lack of congruence between nuclear and mitochondrial markers. While I found eight divergent lineages geographically restricted within Cx. annulirostris and Cx. palpalis at the mitochondrial level, only four lineages appeared at the nuclear level. Two lineages appeared consistent using both markers: Cx. palpalis S-AUS and CX.annulirostris PNG1. Notably, the southern limit of the Cx. annulirostris PNG1 lineage coincides exactly with the current southern limit of JEV activity in Australasia suggesting that biological variation in this lineage may be the key to why JEV has not yet established yet on mainland Australia. I assessed the discrimination power of COl barcoding for Culex mosquitoes and conclude that DNA barcoding using COl may actually overestimate the diversity of Culex mosquitoes in Australasia and should be applied cautiously with support from nuclear DNA, such as the polymorphic ace-2 gene. Finally I designed and developed a PCR-based diagnostic tool to discriminate Cx. annulirostris PNG1 from its morphologically identical relative. This tool will greatly assist identification and surveillance of Cx. annulirostris PNG1 into Australia mainland in the future as it may move south into Australia as a consequence of climate change or population expansion.