Flaviviruses are assembled to bud into the lumen of the endoplasmic reticulum (ER) and are secreted through the vesicle transport pathway, but the details of the molecular mechanism of virion assembly remain largely unknown. In this study, a highly conserved region in the prM protein was identified among flaviviruses. In the subviral particle (SP) system of tick-borne encephalitis virus (TBEV) and Japanese encephalitis virus, secretion of SPs was impaired by a mutation in the conserved region in the prM protein. Viral proteins were sparse in the Golgi complex and accumulated in the ER. Ultrastructural analysis revealed that long filamentous structures, rather than spherical SPs, were observed in the lumen of the ER as a result of the mutation. The production of infectious virions derived from infectious cDNA of TBEV was also reduced by mutations in the conserved region. Molecular modelling analysis suggested that the conserved region is important for the association of prM-envelope protein heterodimers in the formation of a spike of immature virion. These results are the first demonstration that the conserved region in the prM protein is a molecular determinant for the flavivirus assembly process.