A phage display peptide library targeting the nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) strain CH-1a was generated and used for epitope mapping. After 3 rounds of biopanning with the monoclonal antibody (MAb) N3H2 directed against the N protein, 3 positive phages were screened and sequenced. These phages share a consensus sequence, IQTAFNQGA, which corresponds to the amino acid (AA) 79-87 segment of the CH-1a N protein. A small DNA fragment coding for IQTAFNQGA was expressed as a fusion product, and reacted to N3H2 in Western blots and indirect ELISA. Four truncated peptides (IQTAFNQG, IQTAFNQ, QTAFNQGA, and TAFNQGA) expressed as GST fusion products failed to react with N3H2. The sequences around the N3H2-binding site among the N proteins of 57 PRRSV strains were compared. Our results indicate that the IQTAFNQGA motif is highly conserved among North American and European isolates. We concluded that the precisely defined nona-peptide epitope is a novel conserved Linear B cell epitope on the N protein of PRRSV.