A polymerase chain reaction (PCR) assay was developed to detect bovine herpesvirus 4 (BHV4) glycoprotein B (gB) DNA, and a nested-PCR assay was modified for the detection of BHV4 thymidine kinase (TK) DNA in bovine milk samples. To identify false-negative PCR results, internal control templates were constructed, added to milk samples, and co-amplified with viral DNA using the same primers for both templates. Specificity, sensitivity, and reproducibility of the two PCR assays were examined. In both PCR assays, all 31 BHV4 strains examined were scored positive, whereas 14 unrelated viruses scored negative. Sensitivity studies showed that two-ten copies of BHV4 DNA were detectable by the gB-PCR, while one-three copies could be detected by the TK-PCR. For the detection of BHV4 in milk samples, the gB-PCR amplification was found to be ten-times, and the TK-PCR was found to be 55-times more sensitive than virus isolation. BHV4 DNA was detected by gB-PCR and TK-PCR in 93 and 95%, respectively, of 61 milk samples collected from cows infected intramammarily with BHV4, while only 61% were positive by virus isolation. Four out of 48 cows with clinical mastitis were positive for BHV4-gB and BHV4-TK DNA, whereas no BHV4 DNA was detected in milk from control cows. Considerable agreement was seen between the results of the two PCR assays, and both methods were considered as rapid and reliable tests for the screening of BHV4 DNA in bovine milk. The less laborious gB-PCR might be the recommended test of choice for screening large amounts of milk samples for the presence of BHV4.