Japanese encephalitis (JE) is the most important cause of epidemic encephalitis worldwide. JE exists in a zoonotic transmission cycle between Animals/water birds and human by Culex mosquitoes. Assam is one of the highly endemic states for JE sharing approximately 46.95% of total Japanese encephalitis (JE) positive cases reported from the country in 2014.The present study was carried out with the objective of molecular characterization and pathological alterations of JE infection in pig population of Assam. 247 sera samples of pig were screened from Kamrup, Jorhat, Lakhimpur and Goal para and 11.33% of these sera samples showed positive antibody against JEV. The envelope protein gene of JE virus was detected by RT-PCR in 19 blood samples and 5 tissue samples of pigs. The phylogenetic analysis of the JE positive samples belonged to the genotype GIII and was found to be closely related to the human isolates of JEV from Malda (West Bengal), Nadia (West Bengal) and Vellore, India. Hence, suggesting a possible mode of JEV transmission to the mosquito vector and humans inhabiting in the pig rearing areas of Assam.