The functionality of RNA is fully dependent on its structure . For the influenza A virus (IAV), there are confirmed structural motifs mediating processes which are important for the viral replication cycle, including genome assembly and viral packaging . Although the RNA of strains originating from distant IAV subtypes might fold differently, some structural motifs are conserved, and thus, are functionally important . Nowadays, NGS-based structure modeling is a source of new in vivo data helping to understand RNA biology . However, for accurate modeling of in vivo RNA structures, these high-throughput methods should be supported with other analyses facilitating data interpretation . In vitro RNA structural models complement such approaches and offer RNA structures based on experimental data obtained in a simplified environment, which are needed for proper optimization and analysis . Herein, we present the secondary structure of the influenza A virus segment 5 vRNA of A/California/04/2009 (H1N1) strain, based on experimental data from DMS chemical mapping and SHAPE using NMIA, supported by base-pairing probability calculations and bioinformatic analyses . A comparison of the available vRNA5 structures among distant IAV strains revealed that a number of motifs present in the A/California/04/2009 (H1N1) vRNA5 model are highly conserved despite sequence differences, located within previously identified packaging signals, and the formation of which in in virio conditions has been confirmed . These results support functional roles of the RNA secondary structure motifs, which may serve as candidates for universal RNA-targeting inhibitory methods.