The H10 avian influenza viruses (AIV) have been detected in both birds and mammals. Recently, the cases of human infection with H10N8 and H10N3 in China pose high risk to public health. However, the antigenic sites in hemagglutinin (HA) of H10 are poorly understood. In this study, 3 monoclonal antibodies (MAb), designated as 1F4, 6B3 and 6G12, against the HA protein of the H10N3 strain A/chicken/Taizhou/498/2021(H10N3) (TZ498), were first generated. All of these MAb could effectively inhibit TZ498 in haemagglutination inhibition assay and microneutralization assay. Four novel antigenic sites at positions 135, 208, 227, and 266 (H10 numbering) were identified in the HA of TZ498 through escape mutants selected by these 3 MAb. Moreover, natural mutations at positions 135 and 227 were found in the H10 field strains. All these not only provide novel insights into the molecular markers for monitoring the antigenic variation of H10 but also be helpful for developing efficient control strategies against H10.