We have used a set of synthetic overlapping peptides encompassing the entire heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A) to map, in two mouse strains (BALB/c, H2d, and SJL, H2S), the regions on the H-chain recognized by Abs in the last bleed of non-protective anti-BoNT/A antisera and in the bleed of protective antisera immediately following it in the bleeding schedule. Although the protective antisera bound slightly higher amounts of total (IgG+IgM) Abs, non-protective and protective BALB/c antisera showed similar peptide-binding profiles involving peptides N6/N7, N25, C2/C3, C9/C10/C11, C15, C18, C24, C30, and C31 and, at lower amounts of bound Abs, peptides N19, C6/C7, and C28. IgG+IgM antibodies of the protective SJL antisera recognized peptides N5, N22, and C21, and these peptides were only slightly recognized (N22, C21) or unrecognized (N5) by the non-protective antisera. Additionally, peptides N7/N8, N25, C11, C15, and less so N27/N28 bound two-fold or more Abs from the SJL protective antisera than the non-protective antisera. The Abs bound to peptides C4 and C29 were of relatively lower affinity. Peptides C2/C3, C7, C18/C19, C24, C30, and C31 bound higher amounts of Abs in the SJL protective versus the non-protective antisera, but the differences were less than double. We also mapped the binding profiles of the IgG Abs in these sera. BALB/c and SJL had 13-36-fold higher of IgG Abs that bound to BoNT/A in the protective antisera relative to non-protective antisera. The IgG Abs in the protective antisera of each mouse haplotype bound to the same peptides that bound total Abs in the correlate antiserum. But in both mouse strains, the non-protective Abs showed little or no IgG Abs that bound to these peptides. In the SJL haplotype, the IgG response to peptide N5 was transient, appearing strongly in early protective Abs and disappearing by day 70. It is not clear whether the response to region N5 plays a role in initiating and contributing to the protective activity of the toxin in the SJL strain in the early stages but is not needed in later hyperimmune stages of the Ab response. It is concluded that the switch in BALB/c and SJL mice from non-protective to protective Abs is not associated with major changes in the epitope-recognition profiles. Although some slight differences between non-protective and protective antisera appeared in their levels of Abs that were bound by some peptides, these differences are not sufficient to explain differences in the protection properties. Protection was mostly associated with the immunoglobulin class of the antibodies. IgM antibodies were non-protective, while IgG Abs produced after the switch were protective.