Bovine herpesvirus 5 (BoHV-5) and Bovine herpesvirus 1 (BoHV-1) are members of the family Herpesviridae, subfamily Alphaherpesvirinae. Although BoHV-1 and BoHV-5 are closely related viruses (their genomes share 85% similarity), they exhibit some important differences at the genetic level which may explain the differences in their pathogenicity and epidemiological characteristics. Bovine herpesvirus 5 is a neurovirulent virus that causes fatal meningoencephalitis in calves and has been described in Australia, Argentina, United States and Brazil. BoHV-1 causes infectious bovine rhinotracheitis (IBR) and is reported around the world. The aim of this study was to establish the use of random amplified polymorphic DNA (RAPD) followed by phylogenetic analysis to characterize field samples of bovine herpesviruses. This technique was used to study the relationship of Brazilian samples of bovine encephalitis virus isolated in semen and the central nervous system (CNS), including BoHV-1.1 and BoHV-1.2 reference subtype virus for comparison. The samples tested could be clearly separated into BoHV-1.1, BoHV-1.2 and BoHV-5 by RAPD using two different primers. The results showed that RAPD analysis can be used as a diagnostic as well as a molecular epidemiological tool for herpesviruses.