In order to establish a detection method suitable for different strains of OsHV-1,the primers were designed based on the nuclei acid sequence of the conserved DNA polymerase of OsHV-1. A nested PCR detection method (P-nPCR) was established by optimization of the annealing temperatures of the primers and protocols of PCR. Then, both P-nPCR and C-nPCR were employed to test the infection status of the samples collected from different years and hosts. Our results indicated that the detection limits of the P-nPCR detection method was about 100 copies/μL of recombinant plasmid containing OsHV-1 genes. P-nPCR was more specific than C-nPCR in the detection of different variants of OsHV-1, and resulted in a higher prevalence of OsHV-1 for the same samples. In conclusion, a P-nPCR detection method was developed to detect different variants of OsHV-1 infection. The high specificity of P-nPCR to OsHV-1 ensured that different variants of OsHV-1 could be detected as early as possible, which will provide reliable technical support for the detection and epidemiology studies of OsHV-1.