Influenza A virus (IAV) is a respiratory virus that causes epidemics and pandemics. Knowledge of IAV RNA secondary structure in vivo is crucial for a better understanding of virus biology. Moreover, it is a fundament for the development of new RNA-targeting antivirals. Chemical RNA mapping using selective 2´-hydroxyl acylation analyzed by primer extension (SHAPE) coupled with Mutational Profiling (MaP) allows for the thorough examination of secondary structures in low-abundance RNAs in their biological context. So far, the method has been used for analyzing the RNA secondary structures of several viruses including SARS-CoV-2 in virio and in cellulo. Here, we used SHAPE-MaP and dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) for genome-wide secondary structure analysis of viral RNA (vRNA) of the pandemic influenza A/California/04/2009 (H1N1) strain in both in virio and in cellulo environments. Experimental data allowed the prediction of the secondary structures of all eight vRNA segments in virio and, for the first time, the structures of vRNA5, 7, and 8 in cellulo. We conducted a comprehensive structural analysis of the proposed vRNA structures to reveal the motifs predicted with the highest accuracy. We also performed a base-pairs conservation analysis of the predicted vRNA structures and revealed many highly conserved vRNA motifs among the IAVs. The structural motifs presented herein are potential candidates for new IAV antiviral strategies.