West Nile virus (WNV) causes an acute infection that is usually cleared by an effective immune response after several days of viremia. However, a recent study detected WNV RNA in the urine of 5 of 25 persons (20%) tested several years after their initial acute WNV disease. We evaluated an established cohort of 40 persons >6 years after initial infection with WNV. Urine collected from all participants tested negative for WNV RNA by reverse-transcription polymerase chain reaction and transcription-mediated amplification. Prospective studies are needed to determine if and for how long WNV persists in urine following WNV disease.