Type A and type B influenza viruses (FluA and FluB) are two major human pathogens that share common structural and functional features. FluA and FluB could reassort within each type but never between them. Here, we bioinformatically analyzed all promoter sequences of FluA and FluB and confirmed the presence of the type-specific promoter elements. We then studied the promoter elements with cell-based in vivo assays and an in vitro replication initiation assay. Our results identified, for the first time, that a type-specific promoter element - the nucleotide at position 5 in the 3´ end of the vRNA, plays key role(s) in modulating polymerase activity in a type-specific manner. Interestingly, swapping the promoter element between FluA and FluB recombinant viruses showed different tolerances: the replacement of FluA-specific U5 with FluB-specific C5 in A/WSN virus could be immediately reverted to U5 after 2-3 passages; while the replacement of FluB-specific C5 with FluA-specific U5 in B/Yamagata virus could be maintained but with significantly reduced replication efficiency. Therefore, our finding indicates that the nucleotide variation at position 5 in the 3´ end of the vRNA promoter between FluA and FluB contributes to their RNP incompatibilities, which may shed new light on understanding the mechanisms of intertypic exclusion of reassortment between FluA and FluB.IMPORTANCE Genetic reassortment of influenza virus plays a key role in the virus evolution and the emergence of pandemic strains. The reassortment occurs extensively within either FluA or FluB viruses but never between them. Here, we bioinformatically compared available promoter sequences of FluA and FluB and confirmed the presence of the type-specific promoter elements. Our in vivo and in vitro mutagenesis studies showed that a type-specific promoter element - the nucleotide at position 5 in the 3´ end of vRNA promoters plays key roles in modulating polymerase activity. Interestingly, FluA and FluB showed different tolerances upon the key promoter element swapping in the context of virus infections. We concluded that the nucleotide at position 5 in the 3´ end of the vRNA promoters of FluA and FluB is a critical type-specific determinate. This work has implications for further elucidating the mechanisms of the intertypic exclusion of reassortment between FluA and FluB viruses.