The aim of this study was to evaluate the sensitivity of conventional Polymerase Chain Reaction (PCR) assay in comparison to real time PCR assays for detection of BoHV-1 in bovine semen and seminal plasma. Tenfold serial dilutions of BoHV-1 strain were prepared in one ml aliquots of both minimal essential medium (MEM) and negative bovine semen. The dilutions started from 10^{7 to} 10^-4 to detect detection limits of both PCR and real time PCR assays. Detection limits of real time PCR assay were 10^-1 TCID₅₀/ml and 10² TCID₅₀/ml when PCR performed on viral DNA extracted from tenfold dilutions of BoHV-1 in MEM and artificially spiked semen, respectively. When seminal plasma of the same dilutions of spiked semen was used for DNA extraction, the PCR detection limit became 1 TCID₅₀/ ml. Considering conventional PCR, detection limits were 1 TCID50/ml when PCR performed on viral DNA extracted from tenfold dilutions of BoHV-1 in MEM and artificially spiked semen as shown by the presence of a DNA band of the expected size on ethidium bromide-stained agarose gels, respectively. On the other hand, the PCR detection limit was 10 TCID₅₀/ ml when seminal plasma of the same dilutions of spiked semen was used. In conclusion, conventional PCR assay performed in this study provide a satisfactory results for detection of BoHV-1 in bovine semen in comparison to real time PCR and other reported PCR assays. Moreover, the use of seminal plasma instead of whole semen can be better alternative increasing the sensitivity of detection.