Vaccination is the primary strategy for influenza prevention and control. Yet egg-based vaccines, the predominant production platform, have several disadvantages including the emergence of viral antigenic variants that can be induced during egg passage. These limitations have prompted development of cell-based vaccines which themselves are not without issue. Most importantly, vaccine seed viruses often do not grow efficiently in mammalian cell lines. Here we aimed to identify novel high-yield signatures for influenza viruses in continuous Madin-Darby canine kidney (MDCK) and Vero cells. Using influenza A(H1N1)pdm09 virus as the testing platform and an integrating error-prone PCR strategy, we identified a Y161F mutation in the hemagglutinin (HA) that not only enhanced the infectivity of the resultant virus by more than 300-fold, but also increased its thermostability without changing its original antigenic properties. Vaccine produced from the Y161F mutant fully protected mice against wild-type A(H1N1)pdm09 lethal challenge. Compared with A(H1N1)pdm09, the Y161F mutant had significantly higher avidity for avian-like and human-like receptor analogs. Of note, introduction of the Y161F mutation into the HA of seasonal H3N2 influenza A virus (IAV) and canine H3N8 IAV also increased yields and thermostability in MDCK cells and chicken embryotic eggs. Thus, residue F161 plays an important role in determining viral growth and thermostability, which could be harnessed to optimize IAV vaccine seed viruses.IMPORTANCE Although a promising complement to current egg-based influenza vaccines, cell-based vaccines have one big challenge - high-yield vaccine seeds for production. In this study, we identified a molecular signature- Y161F in hemagglutinin (HA) that resulted in increased virus growth in Madin-Darby canine kidney and Vero cells, two commonly used cell lines in influenza vaccine manufacturing. This Y161F mutation not only increased HA thermostability, but also enhanced its binding affinity to α2,6 and α2,3-linked Neu5Ac. These results suggest that a vaccine strain bearing the Y161F mutation in HA could potentially increase vaccine yields in mammalian cell culture systems.