This report describes West Nile virus (WNV)–associated mortality in captive lesser scaup (Aythya affinis) ducklings that occurred in Saskatchewan, Canada, in July and August 2007. There were no clinical signs or gross necropsy findings suggestive of the cause of death; however, microscopic lesions were consistent with WNV infection, including nonsuppurative encephalitis and myocardial, pancreatic, and splenic necrosis. Necrosis of the thymus and thyroid was also observed in some birds, which has not previously been reported in association with WNV infection. Immunohistochemistry revealed WNV antigen in multiple tissues, including thymus and thyroid, and reverse transcription polymerase chain reaction resulted in the identification of WNV gene sequence in all of the ducklings that were tested. This outbreak is of interest because waterfowl (Anseriformes) are not thought to be particularly susceptible to WNV, and there is little information about WNV infection in prefledging birds. The apparent susceptibility of lesser scaup to WNV demonstrated in this study may have implications for declining lesser scaup populations in the wild.