Background <p>West Nile virus (WNV) is an emerging pathogen in Europe with an unprecedented northward expansion. In Romania, the virus has been endemic for at least three decades, and human infection cases are registered yearly, sometimes culminating in outbreak events. Bucharest, the capital city of Romania, is the largest urban area of the country, encompasses a multitude of mosquito and bird habitats, and has been continuously reported as an active hotspot for WNV transmission. The circulation of lineage 2 has been documented in Bucharest since 2010 in mosquito and human populations. Our study aimed to provide up-to-date information on WNV isolates circulating among mosquito vectors collected in Bucharest.</p> Methods <p>Mosquito collection was performed during the 2024 and 2025 WNV transmission seasons at ten sampling sites. WNV was detected in mosquito pools by real-time PCR, and partial and complete genome sequences were obtained and used for inferring phylogeny.</p> Results <p>In both study years, WNV isolates belonging to cluster B of lineage 2a were detected in <i>Culex pipiens</i> s.l. mosquito pools. Phylogenetic analysis indicated the presence of multiple isolate clusters containing sequences previously obtained from samples collected in Bucharest or elsewhere in Romania, as well as from other European countries. The high genetic diversity of WNV lineage 2a in the studied area is sustained by variations observed between sequences obtained from different sites, between sequences from the same site but in different years, and even between sequences from the same site in the same year.</p> Conclusions <p>Close genetic relatedness between the WNV isolates depicted in this study and viruses from European areas with intense WNV circulation in recent seasons, such as Greece, Hungary, Italy, and Russia, or from regions where the virus was recently identified, such as Poland, was demonstrated. A clear trend toward cluster replacement between the two study years was shown.</p> Graphical Abstract <p></p>