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Vector-borne pathogens cause many human infectious diseases and are responsible for high mortality and morbidity throughout the world. They can also cause livestock epidemics with dramatic social and economic consequences. Due to its high costs, vector-borne disease surveillance is often limited to current threats, and the investigation of emerging pathogens typically occurs after the reports of clinical cases. Here, we use high-throughput sequencing to detect and identify a wide range of parasites and viruses carried by mosquitoes from Cambodia, Guinea, Mali and the USA. We apply this approach to individual Anopheles mosquitoes as well as pools of mosquitoes captured in traps; and compare the outcomes of this assay when applied to DNA or RNA. We identified known human and animal pathogens and mosquito parasites belonging to a wide range of taxa, as well as DNA sequences from previously uncharacterized organisms. Our results also revealed that analysis of the content of an entire trap could be an efficient approach to monitor and identify rare vector-borne pathogens in large surveillance studies. Overall, we describe a high-throughput and easy-to-customize assay to screen for a wide range of pathogens and efficiently complement current vector-borne disease surveillance approaches., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)