Nucleic acid testing (NAT) of blood donors provides opportunities for identifying West Nile virus (WNV)-infected persons before symptoms develop and for characterizing subsequent illness. From June 2003 through 2008, the American Red Cross performed follow‐up interviews with and additional laboratory testing for 1436 donors whose donations had initial test results that were reactive for WNV RNA; 821 of the donors were subsequently confirmed to have WNV infection, and the remainder were unconfirmed or determined to have false‐positive results. Symptoms attributed to WNV infection were determined by comparing symptom frequency among 576 donors identified with early WNV infection (immunoglobulin M antibody negative) and those with unconfirmed infection. We estimate that 26% of WNV‐infected persons become symptomatic, defined by the presence of at least 3 of 8 indicator symptoms. Nearly one‐half of symptomatic persons sought medical care; only 5% received a diagnosis of WNV infection. Female subjects and persons with higher viral loads detected in the index donation were more likely than other subjects to develop symptoms.