There are two genotypes among bovine viral diarrhoea viruses (BVDV), BVDV1 and BVDV2. Within the BVDV1 genotype there are two distinct subgenotypes, BVD1a and BVD1b. Serology and monoclonal antibody binding are used to differentiate BVDV from classical swine fever virus (CSFV) and border disease virus (BDV), the other members of the Pestivirus genus. These techniques are less useful in the differentiation and segregation of viruses within the BVDV species. In this study, differential polymerase chain reaction (PCR) amplification has been evaluated as a tool for segregating BVDV isolates into genotypes and subgenotypes. Polymerase chain reaction primers were selected based on the comparison of 5' untranslated region sequences from CSVF, BDV, BVDV1a, BVDV1b and BVDV2. Differential PCR tests were validated using 345 viruses isolated from cattle and small ruminants that had previously been segregated into genotypes and subgenotypes. There was 100% correlation between segregation by differential PCR and the previous segregation of these viral isolates.