question: BACKGROUND: In early 2020, a novel H9N2 AIV immune escape variant emerged in South China and rapidly spread throughout mainland China. The effectiveness of the current H9N2 vaccine is being challenged by emerging immune escape strains. Assessing key amino acid substitutions that contribute to antigenic drift and immune escape in the HA gene of circulating strains is critical for understanding virus evolution and in selecting more effective vaccine components. METHODS: In this study, a representative immune escape strain, A/chicken/Fujian/11/2020 (FJ/20), differed from current H9N2 vaccine strain, A/chicken/Anhui/LH99/2017 (AH/17) by 18 amino acids in the head domain in HA protein. To investigate the molecular determinants of antigenic drift of FJ/20, a panel of mutants were generated by reverse genetics including specific amino acids changes in the HA genes of FJ/20 and AH/17. The antigenic effect of the substitutions was evaluated by hemagglutination inhibition (HI) assay and antigenic cartography. RESULTS: Fujian-like H9N2 viruses had changed antigenicity significantly, having mutated into an antigenically distinct sub-clade. Relative to the titers of the vaccine virus AH/17, the escape strain FJ/20 saw a 16-fold reduction in HI titer against antiserum elicited by AH/17. Our results showed that seven residue substitutions (D127S, G135D, N145T, R146Q, D179T, R182T and T183N) near the HA receptor binding sites were critical for converting the antigenicity of both AH/17 and FJ/20. Especially, the combined mutations 127D, 135G, 145N, and 146R could be a major factor of antigenic drift in the current immune escape variant FJ/20. The avian influenza A (H9N2) variant virus need further ongoing epidemiological surveillance. CONCLUSIONS: In this study, we evaluated the relative contributions of different combinations of amino acid substitutions in the HA globular head domain of the immune escape strain FJ/20 and the vaccine strain AH/17. Our study provides more insights into the molecular mechanism of the antigenic drift of the H9N2 AIV immune escape strain. This work identified important markers for understanding H9N2 AIV evolution as well as for improving vaccine development and control strategies in poultry. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12985-022-01755-9.