The purpose of this study was to reveal the resistance mechanism of Mycoplasma bovis from Xizang yaks to macrolide antibiotics and provide theoretical basis for clinical medication.In this study,10 strains of Mycoplasma bovis from Xizang yaks were tested for drug sensitivity to macrolide antibiotics using the micromethod,and sensitive strains were induced to be highly resist-ant in vitro.The results showed that all 10 strains of Mycoplasma bovis exhibited varying degrees of resistance to macrolide antibiotics.However,through methylation enzyme and inactivation en-zyme gene detection and analysis,it was found that there was no methylation enzyme encoded by the methylation enzyme gene and no resistance mechanism mediated by macrolide inactivation en-zymes,indicating that there were no mutations in the target gene loci of the sensitive and resistant strains.However,highly resistant strains in vitro have mutations at the domain Ⅱ,L22,and even L4 target gene loci,indicating that if two or more target gene amino acid loci undergo mutations,highly resistant strains can be produced.After testing with active efflux systems,it was found that there were no active efflux systems using macrolide antibiotics as substrates.It can be seen that the strain of Mycoplasma bovis from Xizang yaks is prone to mutation under the pressure of high con-centrations of macrolides,and there are mutations in two or more target gene amino acid sites,which is prone to produce highly resistant strains.