West Nile virus (WNV) infection was diagnosed in 13 gray squirrels (Sciurus carolinensis) and 3 fox squirrels (Sciurus niger) that were observed with neurologic signs before death or found dead. All 16 had gliosis throughout all sections of the brain. Most had lymphoplasmacytic encephalitis or meningoencephalitis, many with admixed neutrophils. Neuronal necrosis and neuronophagia were also prominent features. West Nile virus antigen was demonstrated in the brain, spleen, heart or kidney in 10 of 13 gray squirrels and 3 of 3 fox squirrels by immunohistochemistry. Nucleic acid amplification tests (NAATs) confirmed the presence of WNV in the brain or spinal cord of 10/10 gray squirrels and 1/3 fox squirrels tested. Viral levels were quantified in various tissues of selected gray squirrels, and titers were highest in spleen and brain, with no virus detected in serum. This is the first description of lesions associated with WNV infection in gray and fox squirrels.