Blue jays (Cyanocitta cristata) are an effective indicator species for West Nile virus (WNV) and may be regionally important in surveillance efforts. The sites of WNV replication and sensitivity of virus detection techniques are undefined for blue jays. The objectives of this study were to describe the gross and microscopic pathology associated with natural WNV infection in blue jays, as well as determine the most appropriate tissues to be used for virus isolation, reverse transcription-nested polymerase chain reaction, and immunohistochemistry (IHC) techniques. Blue jays were collected in Georgia, USA, between May and September 2001. Initial screening by virus isolation indicated that 36 of 59 blue jays chosen for evaluation were WNV positive. From this group, 20 positive and five negative birds were chosen to compare virus detection techniques. Six positive and five negative birds were selected for histopathology examination. Splenomegaly and poor body condition were the most consistent gross findings among positive birds. The most consistent histopathologic findings in the tissues of WNV-positive blue jays were mononuclear leukocytosis and epicarditis/myocarditis. Brain, heart, and lung had the highest viral titers, and WNV antigen was most often detected by IHC in heart, kidney, liver, and lung. Reverse transcription-nested polymerase chain reaction proved to be the most sensitive diagnostic test applied in this study irrespective of the tissue type. Brain tissue could be used effectively for both virus isolation and RT-nPCR, and this tissue is simple to remove and process. The success of IHC is highly dependent on tissue selection, and the use of multiple tissues including heart, kidney, liver, or lung is recommended.