Using published primers, detection of Mycoplasma synoviae and strain identification using the vlhA gene sequence was attempted. However, of 21 M. synoviae strains examined, three could not be amplified, so a new reverse primer was designed with a target in the conserved region of the vlhA gene. This allowed all 21 M. synoviae strains, a further nine strains and also material from 11 swab samples from M. synoviae-positive birds, to produce a PCR product, suggesting that the method could also be suitable for clinical specimens. The protocol was then tested on the type strains of M. synoviae and the other 22 recognised avian Mycoplasma species, with amplification of M. synoviae only. Further testing demonstrated that this PCR was equally or more sensitive than other PCR tests used to detect M. synoviae. Subsequent DNA sequence analysis of the PCR product based on percent similarity and evolutionary relationship appeared to be a useful tool for strain differentiation.