AbstractEffective vector and arbovirus surveillance requires timely and accurate screening techniques that can be easily upscaled. Next‐generation sequencing (NGS) is a high‐throughput technology that has the potential to modernize vector surveillance. When combined withDNAbarcoding, it is termed ‘metabarcoding.’ The aim of our study was to establish a metabarcoding protocol to characterize pools of mosquitoes and screen them for virus. Pools contained 100 morphologically identified individuals, including one Ross River virus (RRV) infected mosquito, with three species present at different proportions: 1, 5, 94%. Nucleic acid extracted from both crude homogenate and supernatant was used to amplify a 269‐bp section of the mitochondrialcytochrome c oxidase subunitI (COI) locus. Additionally, a 67‐bp region of theRRVE2 gene was amplified from synthesizedcDNAto screen forRRV. Amplicon sequencing was performed using an Illumina MiSeq, and bioinformatic analysis was performed using aDNAbarcode database of Victorian mosquitoes. Metabarcoding successfully detected all mosquito species andRRVin every positive sample tested. The limits of species detection were also examined by screening a pool of 1000 individuals, successfully identifying the species andRRVfrom a single mosquito. The primers used for amplification, number ofPCRcycles and total number of individuals present all have effects on the quantification of species in mixed bulk samples. Based on the results, a number of recommendations for future metabarcoding studies are presented. Overall, metabarcoding shows great promise for providing a new alternative approach to screening large insect surveillance trap catches.