The porcine epidemic diarrhea virus (PEDV) spike (S) protein is the major target of neutralizing antibodies against PEDV. Here immunodominant neutralizing epitopes of PEDV were identified using a panel of S-specific monoclonal antibodies (mAbs). Ten of eleven S-specific mAbs successfully neutralized PEDV infectivity in vitro. Notably, epitope mapping by peptide ELISAs revealed that nine of these mAbs recognized linear neutralizing epitopes located in the N-terminus of the S2 glycoprotein subunit (amino acids aa 744–759, 747–774 and/or 756–771). Additionally, one mAb recognized a neutralizing epitope located in the C-terminus of S2 (aa 1371–1377), while only one neutralizing mAb reacted against a region of the S1 glycoprotein subunit (aa 499–600). Notably, mAbs that recognized epitopes within the S2 subunit presented the highest neutralizing activity against PEDV. Together these results indicate that the S2 glycoprotein subunit contains major antigenic determinants and, perhaps, the immunodominant neutralizing epitopes of PEDV. •A panel of PEDV S-specific neutralizing antibodies were developed and characterized.•S-specific neutralizing mAbs presented potent neutralizing activity against PEDV in vitro.•Epitope mapping revealed that most neutralizing mAbs target the N-terminus of the S2 protein subunit.•S2 subunit seems to contain immunodominant neutralizing epitopes of PEDV.