High prevalence of arboviruses in select mosquito species in Ontario, Canada

California serogroup (CSG) viruses, West Nile virus (WNV), Cache Valley virus (CVV), and eastern equine encephalitis virus (EEEV), are the main mosquito-borne arboviruses of public health concern in Canada. We report findings from a 2017–2021 mosquito sampling campaign in eastern Ontario, Canada, with the aim to optimise surveillance frameworks using mosquito species and spatiotemporal affinities for these endemic arboviruses. Mosquitoes were collected in diverse land uses of eastern Ontario, Canada, from May to October. Female mosquitoes were pooled by species and screened for arboviruses via RT-qPCR. We detected CSG viruses in 610 pools, WNV in 22, EEEV in one, and CVV in one (12,413 pools; 121,042 mosquitoes). In all years, Ochlerotatus provocans showed the greatest infection rate for CSG viruses (up to 22.15 per 1000 mosquitos), while WNV was found mainly in Culex pipiens/restuans (77.2% of WNV-positive pools). The prevalence of CSG viruses was associated with highly forested and lowly urbanised sites, with peak detections in late spring to early summer. High infection rates in certain mosquito species indicate their potential role as vectors and underscore the need for additional vector competence assays. The observed spatiotemporal associations with arboviral activity, particularly for CSG viruses, emphasise the importance of incorporating vector and reservoir ecology and life-history characteristics into arbovirus surveillance frameworks.