Abstract: Objective To understand the pathogenic spectrum of suspected Japanese encephalitis cases in Shanghai. Methods A total of 93 serum samples and 103 cerebrospinal fluid samples were collected from 145 suspected Japanese encephalitis cases during May-October 2010 to detect viral nucleic acid and IgM antibodies of common encephalitis viruses by using real-time PCR and ELISA. The detected viruses included Japanese encephalitis virus (JEV), enteric virus (EV), cytomegalovirus (CMV), herpes simplex virus (HSV), varicellla-zoster virus (VZV). measles virus (MV), respiratory syncytial virus (RSV), mumps virus (MuV) and West Nile virus (WNV). Results A total of 100 samples were detected to be positive either in viral nucleic acid detection or in IgM antibody detection, the positive rate was 68.97% (100/145). Among these 100 patients, 49 (33.79%) were infected with EV, 22 (15.17%) were infected with HSV, 11 (7.59%) were infected with JEV, 5 (3.45%) were infected with CMV, 3 (2.07%) were infected with MuV, and 10 (6.90%) were co-infected with two or more viruses; No VZV, MV, RSV and WNV were detected. Conclusion The major pathogens in suspected Japanese encephalitis cases were EV, HSV and JEV in Shanghai in 2010.The study Results indicated that the surveillance and differential diagnosis of viral encephalitis should be strengthened even in the high incidence season of Japanese encephalitis to provide clinical information for diagnosis and treatment of the disease as well as scientific evidence for the disease control and prevention.