Abstract: Objective To investigate the genotyping, drug resistance and molecular characteristics of Mycobacterium tuberculosis. Methods A total of 391 Mycobacterium tuberculosis strains were selected from department of thoracic surgery from January to December 2018, and genotyped by spoligotyping method. First-line anti-tuberculosis drugs rifampicin (RFP), ethambutol (EMB), isoniazid (INF) and streptomycin (SM) were used for drug sensitive testing. Results The results of genotyping showed that the Beijing family strains were 339 strains (86.70%), the non-Beijing family were 52 strains (13.30%). Spoligotyping showed that the molecular epidemiology of Beijing family bacteria was only positive with 9 compartments between 35 and 43, while the remaining strains was negative in 1-5 sites between 35 and 43. The 391 strains were genotype ranked in the top three: Beijing type, T1 type and newly type, accounting for 86.70%, 5.12%, and 3.58%, respectively. There was no significant difference in the resistance of Beijing-type Mycobacterium tuberculosis to non-Beijing-type mycobacteria for RFP, EMB, INF and SM (all P>0.05). Conclusion The Mycobacterium tuberculosis gene is polymorphic, while the molecular epidemiology characteristics are mainly in the Beijing family and have no significant correlation with drug resistance.