ABSTRACT The influenza virus neuraminidase H274Y substitution is a highly prevalent amino acid substitution associated with resistance to the most heavily used influenza drug, oseltamivir. Previous structural studies suggest that the group specific 252 residue (Y252 in group 1 and T252 in group 2) might be a key factor underlying H274Y resistance. However, H274Y has only been reported in N1 subtypes, which indicates that there must be additional key residues that determine H274Y resistance. Furthermore, we found that members of NA serotype N3 also possess Y252, raising the key question as to whether or not H274Y resistance may also be possible for some group 2 NAs. Here, we demonstrate that the H274Y substitution results in mild oseltamivir resistance for N3. Comparative structural analysis of N3, N1, and their 274Y variants indicates that the interaction of residue 296 (H in N1 and nonaromatic for other serotypes) with conserved W295 is another important determinant of oseltamivir resistance.