Influenza A viruses (IAVs) infect a wide range of mammalian and bird species and are responsible for seasonal outbreaks and occasional pandemics of great consequences in humans. Studying IAVs requires methods to detect the presence of the virus in infected cells or animal models. Recombinant IAV-expressing fluorescent proteins has allowed monitoring of viral infection in cultured cells and ex vivo in the organs of infected animals. However, fluorescent-expressing IAVs are often attenuated and are not suited for the imaging of infected animals using in vivo imaging systems (IVISs). To overcome this limitation, we generated a recombinant pandemic influenza A/California/04/2009 H1N1 (pH1N1)-expressing nanoluciferase (Nluc) from the non-structural viral segment, hereafter referred to as pH1N1-Nluc. The pH1N1-Nluc replicates efficiently in vitro, with growth kinetics and plaque morphology comparable to wild-type pH1N1 (pH1N1-WT). We used this pH1N1-Nluc to demonstrate its ability to effectively identify neutralizing monoclonal antibodies and antivirals, with neutralization and inhibition results comparable to pH1N1-WT. In mice, pH1N1-Nluc was able to induce similar body weight loss and mortality, and viral titers comparable to pH1N1-WT, results that were also recapitulated in a ferret model of IAV infection. Using IVIS, pH1N1-Nluc enabled non-invasive, real-time tracking of viral infection in vivo and ex vivo following infection of mice with viral titers in tissues comparable to pH1N1-WT. The flexibility of this approach was further demonstrated by the generation of a Nluc-expressing recombinant A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Altogether, our results demonstrate that Nluc-expressing recombinant IAVs represent a valuable tool for in vitro and in vivo studies, including the identification of antivirals and/or neutralizing antibodies, and to assess the protective efficacy of vaccines.