Abstract Chikungunya virus (CHIKV) is a mosquito borne alphavirus that causes massive outbreaks of severe debilitating arthritis in humans (1). Despite CHIKV?s status as a significant emerging disease threat, there are currently no approved vaccines or therapies for CHIKV. Therefore, the development of new vaccines/therapies requires that we understand how this important human pathogen replicates and causes disease. While most studies of CHIKV virulence determinants focus on defining the function of viral proteins, there is a growing appreciation of the role of non-coding RNA structural elements in virus replication and pathogenesis. However, the full range of RNA secondary structures within the CHIKV genome is not known, nor is it understood how these structures affect virus biology or disease pathogenesis. We therefore used selective 2?-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to define the RNA structure of the entire CHIKV genome of a clinical isolate from the ongoing CHIKV epidemic in the Americas. This analysis provided the first comprehensive analysis of RNA structure in an alphavirus genome. We identified high levels of RNA secondary structure distributed throughout the genome, including 23 regions with exceptionally high structural stability. Five of these highly stable structures were previously shown (e.g the 5? CSE, 26S promoter region) to play important roles in the CHIKV lifecycle. The remaining 18 novel RNA structures have not previously been characterized. We also identified three different isoforms of the CHIKV 3? UTR. Each differed significantly in their RNA secondary structure, and these differences correlated with their ability to replicate in mosquito cells or cause disease in a mouse model of CHIKV-induced arthritis. These results demonstrate that the CHIKV genome is highly structured, and lead us to hypothesize that the novel RNA structures we identified play important roles in multiple aspects of the CHIKV lifecycle. In Aim 1 we test whether novel, highly stable RNA structures in the CHIKV genome are important for CHIKV replication and disease pathogenesis. In Aim 2 we define the role of a specific RNA structural element in the viral 3? UTR in regulating CHIKV host range and viral virulence. These studies significantly advance our understanding of how alphavirus RNA structure impacts multiple aspects of CHIKV biology, and provide new insights that can be used to develop safer and more effective CHIKV vaccines and therapies.