Purpose <p>Influenza (flu) is a respiratory illness caused by lung infection with influenza viruses. This study establishes lung [¹⁸F]FDG uptake by PET/CT as an accurate measure of lung inflammation associated with influenza A virus (IAV) H1N1 infection.</p> Procedures <p>Immunocompetent BALB/c mice were infected with a highly lethal dose of influenza A virus (PR8 strain) and intravenously injected with [¹⁸F]FDG. <i>Ex vivo</i> tissue biodistribution was assessed by gamma counting, while <i>in vivo</i> tissue biodistribution was analyzed by VOI analysis of PET/CT images. Disease severity was also investigated by VOI measurements of high-resolution lung CT images. Infection and inflammation were confirmed by immunohistochemical staining; while viral replication and expression of inflammatory&#xa0;proteins (cytokines and chemokines) were measured in lung tissues by qRT-PCR and multiplex ELISA, respectively.</p> Results <p><i>Ex vivo</i> tissue biodistribution of [¹⁸F]FDG revealed that the lungs were the only relevant imaging target in influenza-infected mice. Lung [¹⁸F]FDG uptake on PET/CT images increased with disease severity and exhibited 1.53-fold increase on day 1 and up to 2.63-fold increase on day 6 post-infection compared to pre-infection levels. Lung uptake correlated with the increased production of pro-inflammatory proteins associated with influenza infection.</p> Conclusions <p>Lung [¹⁸F]FDG uptake on PET images is a non-invasive molecular biomarker of influenza-A virus-induced lung inflammation and disease, effectively distinguishing infected from non-infected lungs as early as day 1 post-infection.</p>