Five bulls were inoculated intrapreputially with Bovineherpes virus 1 (BHV 1), in order to compare the relative sensitivity of three polymerase chain reaction (PCR) assays for routine diagnosis of fresh bovine semen for the presence of BHV 1 Semen was collected twice a week up to 107 days post-infection (dpi). To reactivate latent virus, the bulls were treated with dexamethasone from 44 until 48 dpi. All samples were examined before and after cryopreservation treatment using a standard virus isolation (VI) method and three PCR assays: PCR A, PCR B and PCR C. PCR A and PCR C used an internal control plasmid DNA template and PCR B used the split sample method in order to control for false negative results. Of the 149 fresh semen samples that were tested, PCR A detected 45 positive, PCR B detected 39 positive and PCR C detected 66 positive, while virus was isolated from 22 samples. Of the 149 samples treated by cryopreservation, the virus was isolated from 13 samples and PCR C was positive in 21 samples. The results demonstrate that all three PCR assays are more sensitive than virus isolation, particularly during the later phases of infection.