Background: A systemic evaluation of the antigenic breadth of the H7 influenza hemagglutinin (HA) proteins, especially for the viruses isolated after 2016, are limited. The purpose of this study was to investigate the antigenic breadth of major H7 strains with an ultimate aim to discover H7 HA proteins that can elicit protective receptor-blocking antibodies against co-circulating H7 influenza strains. Method: A panel of nine H7 influenza strains were selected from 3,633 H7 HA amino acid sequences identified over the past two decades (2000-2018). The sequences were expressed on the surface of virus like particles (VLPs) and used to vaccinate C57BL/6 mice. Serum samples were collected and tested for hemagglutination-inhibition (HAI) activity. The vaccinated mice were challenged with lethal dose of H7N9 virus, A/Anhui/1/2013. Results: VLPs expressing the H7 HA antigens elicited broadly reactive antibodies each of the other vaccine strains, except the A/Turkey/Italy/589/2000 (Italy/00) H7 HA. The putative N-glycosylation site at antigenic site B was identified as a unique antigenic profile of Italy/00. Introduction of the putative glycosylation site (H7 HA-A169T) significantly altered the antigenic profile of HA from the A/Anhui/1/2013 (H7N9) strain. Conclusion: This study focused the importance of key amino acid mutations that result in severe vaccine mismatches for future H7 epidemics. Future universal influenza vaccine candidates will need to focus on viral variants with these key mutations.