Bat influenza viruses are genetically distant from classical influenza A viruses (IAV) and show distinct functional differences in their surface antigens. Nevertheless, any comparative analysis between bat and classical IAV RNA polymerases or their specific subunits are yet to be performed. In this work, we have identified signature residues present in the bat influenza virus polymerase, which are responsible for its altered fitness in comparison to the classical IAVs. Through comparative sequence and structural analysis, we have identified specific positions in the PB2 subunit of the polymerase, with differential amino acid preferences amongst bat and non-bat IAVs. Functional screening helped us to focus upon the previously uncharacterized PB2-282 residue, which is serine in bat virus but harbors highly conserved glutamic acid in classical IAVs. Introduction of E282S mutation in the human-adapted PB2 (influenza A/H1N1/WSN/1933) drastically reduces polymerase activity and replication efficiency of the virus in human, bat and canine cells. Interestingly, this newly identified PB2-282 resides within an evolutionary conserved "S-E-S" motif, present across different genera of influenza viruses and serves as a key regulator of RNA synthesis activity of the polymerase. In contrast, bat influenza viruses harbor an atypical "S-S-T" motif at the same position of PB2, alteration of which with the human like "S-E-T" motif significantly enhances its (H17N10/Guatemala/164/2009) polymerase activity in human cells. Together our data indicates that the PB2-S282 residue may serve as an inherent restriction element of the bat virus polymerase limiting its activity in other host species. Importance Influenza A viruses are known for their ability to perform cross-species transmission, facilitated by amino acid alterations either in the surface antigen, HA, or in the polymerase subunit PB2. Recent isolation of influenza A-like viruses from bats raised the concern about their epizootic and zoonotic potential. Here we identify a novel species-specific signature present within the influenza virus polymerase that may serve as a key factor in adaptation of influenza viruses from bat to non-bat host species. The PB2-282nd residue, which harbors a highly conserved glutamic acid for influenza viruses across all genera (A, B, C and D), encompasses an atypical serine in case of bat influenza viruses. Our data show that the human-adapted polymerase, harboring bat specific signature (PB2-S282) performs poorly, while bat PB2 protein harboring human specific signature (PB2-E282) shows increased fitness in human cells.