The H7 subtype avian influenza virus (AIV) has been reported to infect not only poultry but also humans. The hemagglutinin (HA) protein is the major surface antigen of AIV and plays an important role in viral infection. In this study, five monoclonal antibodies (mAbs) against the HA protein of H7 virus, 2F8, 3F6, 5C11, 5E2 and 5C12, were produced and characterized. Epitope mapping indicated that 103RESGSS107 was the minimal linear epitope recognized by the mAbs 2F8/3F6/5C11, and the other mAbs, 5E2/5C12, recognized the epitope 103-145aa. The protein sequence alignment of HA indicated that the two epitopes were not found in other subtypes of AIV, and all five mAbs did not cross-react with other subtypes, suggesting these mAbs are specific to H7 virus. The epitope 103RESGSS107 was highly conserved among Eurasian lineage strains of H7 AIV, whereas three amino acid substitutions (E104R, E104?K and E104G) in the epitope occurred in 98.44% of North-American lineage strains. Any of these single mutations led the mutated epitope not to be recognized by mAbs 2F8/3F6/5C11; thus, these mAbs can distinguish between Eurasian and North-American lineages of H7 strains. Furthermore, the mAbs 2F8, 3F6 and 5C11 could be highly blocked with H7-positive serum in blocking assays, revealing that 103RESGSS107 may be a dominant epitope stimulating the production of antibodies during viral infection. These results may facilitate future investigations into the structure and function of HA protein, as well as surveillance and detection of H7 virus. RESEARCH HIGHLIGHTS Five mAbs against HA protein of H7 AIV were generated and characterized Two novel epitopes 103RESGSS107 and 103-145aa were identified The epitope 103RESGSS107 differs between Eurasian and North-American lineage The mAbs 2F8, 3F6 and 5C11 could distinguish two lineages of H7 strains.