Background <p>West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus belonging to the family <i>Flaviviridae</i> and the genus <i>Orthoflavivirus</i>. It is primarily transmitted by Culex spp. mosquitoes and maintained in an enzootic cycle involving avian hosts. Humans and horses are considered incidental or dead-end hosts, as they do not sustain onward transmission due to their low viremia levels, although infections may progress to severe neurological disease and death. In Portugal, WNV has circulated in horses almost annually since its first detection in 2004, suggesting endemic establishment.</p> Results <p>This study provides updated data on the geographical distribution and annual occurrence of WNV infection in clinically affected horses in mainland Portugal between 2022 and 2025, thereby contributing to a broader understanding of WNV dynamics in southern Europe. As viremia is typically low and short-lived in horses, diagnosis relies primarily on serological methods, particularly the detection of IgM antibodies. IgM analyses identified WNV-seropositive horses in all four years, with 3/9 cases (33.3%) testing positive in 2022, 15/30 (50.0%) in 2023, 32/52 (61.5%) in 2024, and 13/38 (34.2%) in 2025. No WNV RNA was detected in serum samples. Based on clinical signs together with serological results, WNV was diagnosed in 63 of the 129 horses presenting neurological signs.</p> Conclusions <p>These findings reinforce the continued circulation of WNV in Portugal and highlight the importance of sustained surveillance in equine populations.</p>