During the 2020 West Nile virus (WNV) transmission season, Greece was the most affected EU Member State. More than one third of human cases occurred in Serres regional unit in northern Greece, which is characterized by the presence of a major wetland (Kerkini lake and Strimon river). A total of 2809 Culex pipiens mosquitoes collected in Serres were grouped into 70 pools and tested for WNV. Ten (14.3%) pools were found positive, and all WNV sequences belonged to the Central European subclade of WNV lineage 2. The first human case occurred in a village nearby the lake, and all following cases occurred across the connected river and its tributaries. Similar distribution presented the sites where WNV-positive mosquitoes were detected. The number of Culex spp. mosquitoes per trap per night was higher in 2020 than in previous years (2017-2019). The spatial and temporal distribution of human cases and WNV-positive mosquitoes in 2020 in Serres regional unit suggest that the upsurge of the virus circulation was probably related with factors that affected the ecosystem of the wetland.