[Background] Mycoplasma synoviae (MS) is an important causative agent infecting chicken, which causes enormous losses to the poultry industry in China. [Objective] This study was designed to evaluate the pathogenicity of three isolates from MS outbreak farms, which could enrich our understanding about pathogenicity of MS isolates from different districts. [Methods] Systemic MS infection was induced experimentally in commercial broiler chickens with recent MS isolates (CHN-WF224-2016, CHN-BZJ2-2015 and CHN-JNB19-2016). The virulence of each strain was evaluated by detecting air sac and foot pad lesions, serologic response, tracheal mucosal thickness and MS isolation rates from the infected chicken at 10 and 21 days post-infection and comparing these results with those obtained from a live attenuated vaccine strain (MS-H) and uninfected controls. [Results] Isolates CHN-BZJ2-2015 and CHN-WF224-2016 induced foot pad lesions and typical infectious synovitis, especially CHN-BZJ2-2015 group. Serologic response and tracheal mucosal thickness measurements at 21 days post infection indicated that CHN-BZJ2-2015 isolate was significantly more virulent than the CHN-JNB19-2016 isolate and MS-H strain (P<0.05). [Conclusion] The variation in virulence of MS isolates emphasizes the importance of active, on-going control and prevention of MS in the chicken flocks of China and accumulates experimental data for vaccine development.