In the present investigation we used native and recombinant TsNTxP to elicit antibodies in three different animal models (mouse, rabbit and sheep). Differences among anti-TsNTxP antibodies were analyzed using sets of overlapping pentadecapeptides of the TsNTxP amino acid sequence and also modified peptides to reveal key residues in antibody-peptide binding. Despite the identification of similar peptides by the antibodies in the C and N-terminal, peculiarities of each system were observed including the level of reactivity and also the number and type of key residues in the continuous epitopes of TsNTxP. In addition, in vitro neutralization assays indicated that sheep are an alternative and efficient model for the production of anti-Tityus serrulatus venom.