Japanese Encephalitis Virus (JEV) is a flavivirus responsible for an important zoonotic, vector‐borne disease included in the OIE list. JEV is endemic in a large area of Asia. In Italy, JEV has been found in dead birds collected in 1997–2000 and in a pool of Culex pipiens mosquitoes collected in 2010. Viral ecology in the inter‐epidemic periods is not known. The aim of this study was to investigate JEV in FFPE archival samples of healthy birds collected in 1997–2000 in Tuscany (Italy) in the same area and a few months after collection of birds resulted infected by JEV. Different samples from 37 young birds and 83 adults were available. Immunohistochemistry detected JEV antigen only in bone marrow samples from 12 young healthy birds. Positive cells were morphologically referable to monocyte–macrophages lineage and were positive for anti‐CD11b in serial sections. Real‐time PCR detected JEV RNA in four of these samples. These results suggest that healthy birds can harbour JEV in bone marrow cells, while no other organs resulted infected. The role of healthy birds in JEV ecology should be better investigated. Surveillance programmes should include sampling of the most appropriate target organs.