The Spanish imperial eagle (Aquila adalberti) is considered to be the most endangered European eagle. The species is an endemic resident in the Southwestern Iberian Peninsula. We used RT-PCR, immunohistochemistry and seroneutralization to test samples from 13 wild and 18 captive eagles. WNV was detected by RT-PCR in tissues and/or oropharyngeal swabs of eight of 10 (80%) imperial eagles analyzed, and both in apparently clinically healthy birds, and in animals that died due to secondary infections but had symptoms/lesions compatible with WNV. Immunohistochemistry detected WNV antigen in Purkinje cells in the cerebellum, epithelial cells of the gizzard and duodenum, perivascular inflammatory cells, and in Kupffer-cells and hepatocytes. Serum antibodies against WNV were detected in a total of five out of 21 imperial eagles (23.8%), including free-living nestlings (two out of nine samples, 22.2%) and captive adult eagles (three out of 12 samples, 25%). Our results evidence WNV circulation among free-living and captive Spanish imperial eagles in South-central Spain, a dry inland region with no previous WNV evidence, throughout 6 consecutive years. They also indicate the need for further research into this important zoonosis in order to better understand its epidemiology in the Mediterranean ecosystem and in order to understand the role of WNV in the population dynamics of the critically endangered Spanish imperial eagle.