Viral RNA structure plays an important role in the replication and translation of many RNA viruses, however its role in viral evolution and emergence is still poorly understood. Alphaviruses are positive sense RNA arthropod-borne viruses that cause serious disease in humans and animals. Most alphaviruses including Venezuelan equine encephalitis virus (VEEV) and Sindbis virus (SINV) continually cycle between arthropod and vertebrate hosts and therefore must continually adapt to replicating in these highly dissimilar hosts. RNA structure has been shown to play a key role in virus-host switching of arboviruses. We have previously shown that 5’ and 3’ untranslated region (UTR) RNA structures that differ amongst pathogenic and attenuated alphaviruses facilitate evasion of the host interferon (IFN) response, and hypothesize that other viral RNA structures contribute to emergence and pathogenesis of alphaviruses, and other +ssRNA viruses. We have developed a computational approach to identify putative novel RNA structures that differ between viruses with distinct properties (e.g. enzootic and epizootic VEEV). Using reverse genetics, molecular, and biochemical approaches, we have validated candidate RNA structures and show that they contribute to cellular tropism and pathogenesis of these viruses. We have also begun applying this approach to identifying RNA structures important for cross-species transmission and emergence of other zoonotic viruses, suggesting a broad role for viral RNA structure in zoonotic emergence.