BackgroundRoss River virus (RRV) is Australia’s most common and widespread mosquito-transmitted arbovirus and is of significant public health concern. With increasing anthropogenic impacts on wildlife and mosquito populations, it is important that we understand how RRV circulates in its endemic hotspots to determine where public health efforts should be directed. Current surveillance methods are effective in locating the virus but do not provide data on the circulation of the virus and its strains within the environment. This study examined the ability to identify single nucleotide polymorphisms (SNPs) within the variable E2/E3 region by generating full-length haplotypes from a range of mosquito trap-derived samples.MethodsA novel tiled primer amplification workflow for amplifying RRV was developed with analysis using Oxford Nanopore Technology’s MinION and a custom ARTIC/InterARTIC bioinformatic protocol. By creating a range of amplicons across the whole genome, fine-scale SNP analysis was enabled by specifically targeting the variable region that was amplified as a single fragment and established haplotypes that informed spatial-temporal variation of RRV in the study site in Victoria.ResultsA bioinformatic and laboratory pipeline was successfully designed and implemented on mosquito whole trap homogenates. Resulting data showed that genotyping could be conducted in real time and that whole trap consensus of the viruses (with major SNPs) could be determined in a timely manner. Minor variants were successfully detected from the variable E2/E3 region of RRV, which allowed haplotype determination within complex mosquito homogenate samples.ConclusionsThe novel bioinformatic and wet laboratory methods developed here will enable fast detection and characterisation of RRV isolates. The concepts presented in this body of work are transferable to other viruses that exist as quasispecies in samples. The ability to detect minor SNPs, and thus haplotype strains, is critically important for understanding the epidemiology of viruses their natural environment.Graphical AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s13071-023-05734-z.