Reporter viruses provide a powerful tool to study infection, yet incorporating a non-essential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus with NanoLuc luciferase inserted into the 5´ end of the PA gene segment grew to titers 10-fold less than wild-type in MDCK cells and in DBA/2 mice and was less virulent. For twelve rounds, we propagated DBA/2 lung samples having the highest ratios of bioluminescence-to-titer. Every three rounds, we compared in vivo replication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino-acid mutations and PB2 non-coding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-like in vivo fitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCE Influenza viruses contribute to 290,000-650,000 deaths globally each year. Infection is studied in mice to learn how it causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene into the virus that causes infected cells to light up, scientists could now see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.