A set of primers (BTV-pr1/2) were selected that hybridized to the VP3 gene of the major North American serotypes of bluetongue virus (BTV). Polymerase chain reaction (PCR) testing yielded positive results from specimens of major North American BTV isolates (serotypes 10, 11, 13 and 17) propagated in Vero cells. In addition, PCR assays were positive from samples of all other BTV serotypes, except BTV-16; however, an alternative primer pair (BTV-prN1/N2) was devised for amplification of this serotype and the major North American BTV serotypes. PCR products were not evident following amplification of related viruses, epizootic haemorrhagic disease virus (EHDV) serotypes 1 or 2, in either PCR test. In addition, slight modification of the nucleic acid extraction method allowed for the amplification of BTV template from ovine and cervine blood, but not from the respective control blood samples. Restriction endonuclease analysis (REA) using AluI and TaqI discriminated the PCR products of BTV serotypes 10, 13 and 11/17. Identification of BTV-11 and -17 was accomplished by PCR product nucleotide sequencing. Thus, using a single gene region (VP3), nucleic acid amplification methods were devised for expeditious serogroup-specific detection of all BTV serotypes and identification of individual North American BTV nucleotypes, which is expected to prove valuable for disease control strategies and retrospective epidemiological analyses.