Objective To investigate the mutation characteristics and influencing factors of ethambutol （EMB） resistance gene of Mycobacterium tuberculosis in Guangxi Zhuang Autonomous region， and to provide evidence for molecular diagnosis and clinical treatment of tuberculosis. Methods A total of 655 strains of Mycobacterium tuberculosis （52 ethambutol resistant strains and 603 ethambutol sensitive strains） were collected continuously from 30 TB drug resistance monitoring sites in Guangxi in 2018-2019， and the mutation characteristics and influencing factors of ethambutol resistant genes were analyzed by whole genome sequencing. Results Among 655 strains of Mycobacterium tuberculosis， 54 strains had ethambutol drug resistance gene mutation， the mutation rate was 8.24% （54/655）. Among 52 EMB-resistant strains detected by proportional method， 21 had gene mutation， the mutation rate was 40.38% （21/52）， and 33 of 603 EMB-sensitive strains had gene mutation， the mutation rate was 5.47% （33/603）. The gene mutation rate in drug-resistant strains was higher than that in sensitive strains （χ ² =77.133， P = 0.000）. The coincidence rate of EMB drug resistance phenotype and gene mutation was 40.38%（21/52）， and the results of the two tests were not highly consistent （Kappa = 0.343， P < 0.001）. The mutant genes of 54 strains were embA， embB and embC， and there were 20 mutant forms， among which 29 were mutated at unit point， accounting for 53.70% （29/54）， and 25 were mutated at joint site， accounting for 46.30% （25/54）. Among the unit point mutations， embB306（35.19%） had the highest mutation proportion， followed by embB497 （5.56%） and embB406 （3.70%）. Among the joint site mutations， embC270+embB378 had the highest mutation proportion （22.22%），The second was embB306+embA-12 （3.70%）. Gender， anti-tuberculosis treatment history， genotype and MDR might be related to EMB gene mutation （χ ² = 9.388， P = 0.004； χ ² = 27.084， P = 0.000； χ ² = 6.671， P = 0.010； χ ² = 68.826， P = 0.000）. Multivariate logistic regression analysis showed that male （OR = 6.150）， retreatment （OR = 2.636） and multidrug resistance （OR = 7.333） may be risk factors for EMB resistance gene mutation， and Beijing genotype may be a protective factor for EMB resistance gene mutation （OR = 0.511）. Conclusion EMB resistance of Mycobacterium tuberculosis is related to embA， embB and embC gene mutations， and the incidence of EMB resistance phenotype is not high. For male， retreatable， MDR-resistant， and non-Beijing genotype TB patients， attention should be paid to the mutation of the EMB resistance gene.