Japanese Encephalitis virus (JEV) can be separated into 5 genotypes (g1 to g5) based on sequence similarity. JEV g5 strains have been rarely isolated, and are poorly characterized. We report here the full characterization of a g5 virus generated using a cDNA-based technology, and its comparison with a widely studied g3 strain. We did not observe any major differences between those viruses when their infectious cycles was studied in various cell linesin vitro. Interestingly, the JEV g5 strain was highly pathogenic when inoculated to BALB/c mice, which are known to be largely resistant to JEV g3 infection. The study of chimeric viruses between JEV g3 and g5 showed that there was a poor viral clearance ofviruses that express JEV g5 structural proteins in BALB/c mice blood, that correlated with viral invasion of the central nervous system, and encephalitis. In addition, using an in vitro model of the blood-brain barrier, we were able to show that JEV g5 does not have an enhanced capacity at entering the central nervous system, when compared to JEV g3. Overall,in addition to providing a first characterization of the understudied JEV g5, our work highlights the importance of sustaining an early viremia inthe development of JEV encephalitis. Genotype 5 viruses are genetically and serologically distinct from other JEV genotypes and can been associated with human encephalitis, which warrants the need for their characterization. In this study, we characterized the in vitro and in vivo properties of a JEV g5 strain, and showed that it was more neuropathogenic in a mouse model than a well-characterized JEV g3 strain. The enhanced virulence of JEV g5 was associated with poor viral clearance but not with enhanced crossing of the blood-brain barrier, thus providing new insights into JEV pathogenesis.