Vaccination against seasonal influenza is considered an effective means of reducing morbidity and mortality. Live-attenuated vaccines offer more protection against influenza than inactivated vaccines as they efficiently induce cellular immunity and provide cross-immunogenicity against various antigenic subtypes. For the production of safer live-attenuated vaccines, it is important to develop a common master donor vaccine strain in which pathogenic revertants are much less likely to appear. In this study, we introduced a single amino acid substitution of Lys471 into the PB1 polymerase subunit of influenza A virus and succeeded in isolating an attenuated mutant virus that exhibits a temperature-sensitive phenotype. The Lys471 residue is located in the polymerase motif D on PB1 and is positioned near the entrance tunnel domain for incoming nucleotide triphosphate. Although 10 viable PB1-Lys471 mutants did not proliferate at 37°C, their variants could replicate at 31°C and 34°C. Moreover, we found that PB1-Lys471Pro variant induces a genetically stable influenza virus phenotype; this mutant virus did not revert to wild-type phenotype from the temperature-sensitive phenotype by serial virus passages. Animal experiments have demonstrated that these PB1 mutant strains work effectively as live-attenuated vaccines. Application of the PB1-Lys471 substitution to a master donor strain is expected to lead to the development of a safer, high-performance, and widely used live-attenuated vaccine with the antigen of circulating influenza viruses.