The rise of Zika Virus (ZIKV) infections associated with neurological disorders has raised public health concerns. Mutations in pre-epidemic strains are hypothesized to be responsible for increased virulence of epidemic strains. We identified virulence-associated mutations in epidemic strains and introduced these mutations into the pre-epidemic MR766 strain, characterizing infection in vitro. Comparison of mutant viruses with pre-epidemic ZIKV suggests genomic regions coevolved to produce strains associated with neurological disease.