Porcine reproductive and respiratory syndrome virus (PRRSV) has caused tremendous economic losses and continues to be a serious problem to the swine industry worldwide. The structure and function of PRRSV nonstructural protein 12 (NSP12) is still unknown. In this study, we produced a monoclonal antibody, named as 1E5, against the NSP12 protein of HP (highly pathogenic) -PRRSV strain HuN4. A series of partially overlapping recombinant NSP12 truncations and synthesized peptides were used to define the epitope recognized by 1E5. We found that 130KANATSMRFH139 is the minimal linear epitope and that it is highly conserved among some HP-PRRSV isolates of type 2 PRRSV, but not the classical isolates of type 2 PRRSV or the isolates of type 1 PRRSV. Therefore, 1E5 can be used to establish a valuable tool to distinguish infections with HP-PRRSV isolates of type 2 PRRSV from the classical isolates of type 2 PRRSV and type 1 PRRSV.