In order to test the virulence of three strains of Mycoplasma bovis（HS2019，HSZ2019 and HSS2019）isolated from an infected farm in Inner Mongolia，an animal regression test was carried out，the number of Mycoplasma in tissue and organs was determined by observing clinical symptoms and pathological changes after virus challenge as well as by real-time fluorescent quantitative PCR，then the virulence of the three strains was analyzed. The results showed that such clinical symptoms as increasing body temperature，cough and dyspnea appeared in experimental cattle after the regression test of the three strains. The lung lesions as well as adhesion between lung and thoracic cavity and other pathological changes could be observed after anatomy，especially HS2019 strain，by which more obvious symptoms and lesions were caused，and the volume of Mycoplasma in tissues was maximum. In conclusion，the three strains of Mycoplasma were all pathogenic，especially HS2019 strain whose virulence was the strongest，which could be used as the preparation strain for vaccine development in the future. By the study，strain resources were provided for the development of vaccines，and evaluation criterion for immune challenge test of Mycoplasma bovis in the future was also provided.