The 349-amino-acid major core protein VP7 of bluetongue virus (BTV) is both the most abundant viral structural protein and the major immunogenic serogroup-reactive viral antigen. Previous studies indicated that a conformation-dependent antigenic site, defined by the VP7-specific monoclonal antibody 20E9/B7/G2(20E9), was accessible from the virus surface and that the binding of the monoclonal antibody to this epitope could be blocked specifically by antisera raised against different serotypes of bluetongue virus, suggesting it is a serogroup-specific immunodominant epitope. Using a combination of three different mapping strategies, we have located the 20E9 binding site at the N-terminus of the molecule, between amino acids 30 and 48. The fine mapping of the 20E9 immunodominant epitope will facilitate structure-function analyses of the major core protein and provide new opportunities to improve existing BTV serodiagnosis methods based on this immunogenic site.