The polymerase chain reaction was used to detect the presence of bluetongue virus (BTV) in a number of clinical and insect samples collected in the Northern Territory of Australia. Sequence analyses of the amplified BTV genes differentiated endemic Australian and exotic viruses. Two potential exotic BTV were detected as a result of PCR analyses of blood from sentinel animals and of the insect vector, Culicoides wadai. The detection of BTV in C wadai was the first direct demonstration of the presence of BTV in this potential vector. This new technology can significantly reduce the time taken for a diagnosis from a clinical sample and increase the amount of useful information obtained on a BTV isolate by using rapid sequencing techniques. Sequence data were used to differentiate between BTV20 isolated in 1975 and two isolates of the same serotype, isolated in 1992, and indicated that the latter were probably a recent incursion into Australia from Indonesia due to their greater VP3 sequence homology to the BTV9 (Java) than to Australian BTV isolates.