Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. The transmission dynamics of JEV is accountable for more than 3 billion people at risk of infection living in approximately 24 endemic countries in the WHO Southeast Asia and Western Pacific regions. JEV is a mosquito-borne Flavivirus of ~40–50 nm in diameter, which is an enveloped RNA virus of ~10,000 base pairs flanked by 3′ and 5′ untranslated regions (UTRs). JEV has been classified into five genotypes (GI–GV), among which the GI has displaced the previous GIII and become the dominant JEV genotype. The genome encodes for a long polypeptide chain of 3432 amino acids with a single ORF. The polypeptide gets co- and posttranslationally processed into three structural capsid (C), envelope (E), and pre-membrane (prM) and seven nonstructural proteins identified as NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5. The structural and nonstructural proteins are involved in the replication and immune escape mechanism during JEV infection. In spite of diverse understanding of the structural aspects of the JEV, still there is a lack of effective antiviral drugs. Therefore, this chapter focuses on the recent understanding of the JEV structure, genome organization, and replication.