An analysis that combined bioinformatics, comparative sequence/structural analysis, and experimental assays has been completed on respiratory syncytial virus (RSV). Both the genomic RNA and its reverse complement were studied using the novel bioinformatics pipeline ScanFold, which predicted 49 regions on RSV RNAs that appear to encode functional secondary structures (based on their unusually stable sequence order). Multiple motifs appear to be conserved between RSV and related virus strains, including one region within the F gene, which had a highly favorable overall prediction metric of a paired secondary structure. This motif was subjected to additional experimental analyses using SHAPE analysis to confirm ScanFold predicted secondary structure. In subsequent analysis, RSV F mRNA transcripts were made by in vitro transcription using T7 polymerase and transcripts which relaxed the predicted secondary structure yielded slightly higher mRNA transcripts and protein expression levels as wildtype F. However, using reverse genetics for comparison of viruses containing wildtype or relaxed F suggested that the predicted secondary structures may be critical for RSV replication in cells. To our knowledge, this is the first study to examine conserved RNA structures across multiple RSV strains and may help identify potential therapeutic targets to inhibit.