H7N9 avian influenza virus (AIV) poses a persistent threat to poultry and public health. Despite widespread vaccination in China, rapid antigenic drift and reassortment necessitate frequent updates of vaccine strains. Phylogenetic analysis of isolates from Chinese provinces (from 2019 to 2023) showed that while surface genes diversified, the internal gene cassette remained conserved yet actively reassorted with other subtypes, suggesting internal gene compatibility may influence vaccine performance. We selected the H7N9 strain A/chicken/Northeast China/19854-6/2019 (E2), which harbors a polybasic Hemagglutinin (HA) cleavage site and predicted dual receptor-binding affinity. To enable safe vaccine development, we modified the HA cleavage site to generate a low-pathogenicity strain (E2-Δ). Using reverse genetics, we constructed three recombinant viruses: E2-Δ (retaining contemporary internal genes), CVI-E2, and CVII-E2 (containing internal genes from commercially used donor strains CVI and CVII, respectively). Inactivated vaccines were evaluated in specific-pathogen-free (SPF) chickens. E2-Δ induced HI antibody titers comparable to those of CVI-E2 and significantly higher than those of CVII-E2, and showed modestly higher cross-reactive HI titers against some recent H7N9 variants in exploratory analyses. All vaccines provided complete homologous protection with reduced viral shedding and no clinical signs in challenge trials. Our findings suggest that internal gene backbone compatibility may influence vaccine immunogenicity. While E2-Δ outperformed CVII-E2, it was comparable to CVI-E2, indicating that certain traditional backbones may still be suitable for H7N9 vaccine development. This approach warrants further validation to support a refined vaccine design strategy for H7N9 and potentially other avian influenza subtypes.