(1)Background: Dipeptidyl Peptidases IV (DPPIVs), present in many organisms, are minor components in the venoms of Hymenoptera, where they have been shown as cross-reactive allergenic molecules. Since the structure of homologous DPPIVs is well characterized, we explain which regions have higher similarity among these proteins and present a comparison including a new Vespa velutina DPPIV sequence. Moreover, two cases of sensitization to DPPIV in wasps- and honeybees-sensitized patients are presented. (2) Methods: Proteomic analyses have been performed on the venom of the Asian Hornet V.velutina, in order to demonstrate the sequence of its DPPIV (putative allergen Vesp v 3). Comparison by alignments and analysis of the three-dimensional structure allow to show a region with higher similarity among Hymenoptera DPPIVs. Besides, ImmunoCAP™ determinations (including specific inhibition experiments), as well as IgE-immunoblotting, demonstrate the presence of Api m 5 and Ves v 3. (3) Results and conclusions: The data presented explain that the similarities among Hymenoptera DPPIVs are most probably localized at the C-terminal region of these enzymes. The clinical cases analyzed demonstrate the presence of this minor component in the preparations used in venom immunotherapy. Moreover, a new DPPIV sequence is published (Accession Number P0DRB8).