Zika virus (ZIKV) strains can be classified into the ancestral African and contemporary Asian lineages, with the latter responsible for recent epidemics associated with neurological conditions. To understand how Asian strains lead to exacerbated disease, a crucial step is identifying genomic variations that affect infectivity and pathogenicity. Here we use two high-throughput sequencing approaches to assess RNA secondary structures and intramolecular RNA-RNA interactions in vivo for the RNA genomes of Asian and African ZIKV lineages. Our analysis identified functional RNA structural elements and a functional long-range intramolecular interaction specific for the Asian epidemic strains. Mutants that disrupt this extended RNA interaction between the 5' UTR and the E protein coding region reduce virus infectivity, which is partially rescued with compensatory mutants, restoring this RNA-RNA interaction. These findings illuminate the structural basis of ZIKV regulation and provide a resource for the discovery of RNA structural elements important for ZIKV infection.