Alphaviruses are positive-sense, single-stranded RNA (+ssRNA) viruses transmitted by arthropod vectors to vertebrate hosts. Given that alphavirus transmission requires replication in multiple tissues in both arthropod and vertebrate hosts, it is crucial that they optimize genome functionality for efficient transmission. One possible adaptation includes RNA modifications, as evidence suggests that RNA modifications are prevalent in Sindbis virus (SINV) RNA, with pseudouridine being the most prevalent. Given that pseudouridine affects cellular RNA function, its prevalence suggests an important role in alphavirus biology. However, the location, function, and the cellular machinery that deposit pseudouridine are not known. In these studies, we identified a conserved host pseudouridine synthase that is proviral for SINV infection in insects and humans. Furthermore, ectopic expression of Nop60B in cell culture increased SINV infectivity and intracellular RNA levels, which is dependent on the catalytic function of Nop60B. We found that this proviral function is conserved in vertebrate host cells as the human ortholog, dyskerin, increases SINV replication. Using Psi-seq, we mapped putative pseudouridine sites within SINV RNA. A silent mutation at one of these sites led to a reduction in SINV replication, indicating a potential functional role. We extended our Psiseq analyses to identify putative pseudouridine sites in SINV and Chikungunya virus (CHIKV) RNA in both mosquito and human host cells, confirming that pseudouridine is widespread in SINV and CHIKV. We identified unique and shares sites in each virus derived from the human and mosquito cell lines. Sites identified in this study coincide iv with previously characterized cis-acting RNA elements. Overall, my findings suggest that pseudouridine and the pseudouridine synthases contribute to alphavirus replication, adding to our broader understanding of viral replication mechanisms