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West Nile virus is a flavivirus transmitted to humans mainly by mosquito bites. Outbreaks are observed in several European countries, and Greece is one of the most affected countries during the recent years. Thessaly was one of the most affected regions in Greece in 2019. A total of 3,025 Culex spp. mosquitoes collected in Thessaly were grouped into 47 pools and tested for West Nile virus (WNV). Eight (17%) pools were found positive. Whole genome sequences were obtained from two positive pools. Phylogenetic analysis showed that the causative strain was an evolutionary variant of the strains circulating in 2018 belonging to the Balkan subgroup of the Central European subclade of WNV lineage 2.