Bovine herpesvirus 5 (BoHV-5) is an important pathogen of the central nervous system and has already been described in the genital tract of cattle and in semen. This virus is responsible for sporadic epizootics of fatal meningoencephalitis of calves. The objective of the present study was the identification and characterization of BoHV-5 in semen samples from bulls for the first time in Iran. DNA was extracted from bull semen samples, and the glycoprotein D (gD) gene of BoHV-5 and also the thymidine kinase (tK) gene of bovine herpesvirus 1 (BoHV-1) were amplified by PCR assay. The results showed a high prevalence of BoHV-5 (73.2 %) and BoHV-1 (25.89 %) in Iranian bull semen samples. In addition, in order to identify and compare BoHV-5 isolated from Iranian bulls with other isolates from all over the world, the gD gene of this virus was cloned and sequenced. A BLAST search showed that the sequence of the gD gene of BoHV-5 from Iran was 99 % identical to other sequences in the GenBank database. The present study indicated that semen samples are important transmission sources of BoHV-5 virus in Iranian bulls.