The proportion of laboratory-confirmed Japanese encephalitis (JE) virus (JEV) infections was compared to the number of JE cases reported on the basis of seasonality and the clinical symptoms of hospitalized patients in Guizhou Province, China, between April and November 2006. Of the 1,837 patients with reported JE, 1,382 patients in nine prefectures were investigated. JE was confirmed in 1,210 of 1,382 (87.6%) patients by a JEV-specific immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA), heminested reverse transcriptase PCR, and virus isolation. Two strains of JEV belonging to genotype 1 were isolated. Other viral pathogens responsible for encephalitis, including echovirus, mumps virus, herpes simplex virus, and cytomegalovirus, were identified in 67 of 172 (38.9%) JE-negative cases. On the basis of the distribution of the laboratory-confirmed JE cases from different hospitals according to the Chinese administrative division, which included hospitals at the provincial, city, county, and township levels, county hospitals detected the highest number of JE cases (81.8%), whereas township hospitals detected the smallest number of JE cases (1.4%). Provincial and city hospitals had the highest and lowest rates of accuracy of providing a clinical diagnosis of JE, as confirmed by laboratory testing (91.8% and 76.7%, respectively). This study demonstrates that laboratory confirmation improves the accuracy of diagnosis of JE and that an enhanced laboratory capacity is critical for JE surveillance as well as the identification of other pathogens that cause encephalitic syndromes with clinical symptoms similar to those caused by JEV infection.