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Mycoplasma synoviaeinfections result in significant economic losses in the chicken and turkey industries. A commercially available live temperature-sensitive (ts+) vaccine strain MS-H has been found to be effective in controllingM. synoviaeinfections in commercial layer and broiler breeder farms in various countries, including Australia. Detection and differentiation of MS-H from field strains (ts−) and fromts−MS-H reisolates in vaccinated flocks is vital in routine flock status monitoring. At present microtitration is the only available technique to determine thetsphenotype ofM. synoviae. This technique is time consuming and not amenable to automation. In the present study, a quantitative real-time polymerase chain reaction (Q-PCR) was combined with simultaneous culturing ofM. synoviaeat two different temperatures (33°C and 39.5°C) to determine thetsphenotype of 22 AustralianM. synoviaestrains/isolates. TheM. synoviaetype strain WVU-1853 was also included for comparison. A ratio of the copy numbers of the variable lipoprotein haemagglutinin (vlhA) gene at the two temperatures was calculated and a cut-off value was determined and used to delineate thetsphenotype. In allM. synoviaestrains/isolates tested in this study, thetsphenotype determined using Q-PCR was in agreement with that determined using conventional microtitration. Combination of Q-PCR with differential growth at two different temperatures is a rapid, reliable and accurate technique that could be used as an effective tool in laboratories actively involved intsphenotyping ofM. synoviaestrains/isolates. [ABSTRACT FROM PUBLISHER]