In the present study, we report the detection, isolation, molecular characterisation and phylogenetic analysis of bovine herpesvirus type 1 (BoHV-1) in nasal swabs, sera and tissue samples derived from naturally infected cattle. In the spring of 2008, clinical symptoms of the respiratory disease were observed in three Croatian dairy herds. Nasal swabs and sera were collected on the 5th and 27th days after appearance of symptoms. A tissue sample (lungs) from an eight week old calf was also collected. Specific antibodies against BoHV-1 were not found in sera samples collected after the first sampling, but all sera samples were positive after the second sampling. The cytopathic effect on the bovine embryo kidney (BEK) cells was visible after 72 hours for the nasal swab samples, and after 48 hours for the lung tissue sample. Sensitive PCR assays specific for BHV-1 glycoproteins gB and gC were also performed. All nasal swab samples and the lung sample were positive with both sets of PCR primers. The gC PCR product obtained from one sample from each farm as well as from the lung sample DNA were sequenced. Phylogenetic analysis of gC gene sequence clustered our isolates with BoHV subtype 1.1.