Objective To study the polymorphism and epidemic characteristics of the Beijing genotype strains of Mycobacterium tuberculosis (Mtb) in Yichang area, reveal the molecular epidemiological characteristics of tuberculosis in this area, and provide scientific evidence for the prevention and treatment of tuberculosis. Methods 298 strains of Mtb with complete case information from January 2018 to December 2019 were selected for the identification of human Mtb, Beijing genotype strains were identified by multiplex polymerase chain reaction (DTM-PCR) which amplifying RD105 deletion fragment; polymorphism of Beijing genotype strains was analyzed by optimized 9-locus variable-number tandem repeat technique. Results Among 298 strains of Mtb, 260 strains were human Mtb, DTM-PCR identification found that 140 strains (53.85%) were Beijing genotype and 120 strains (46.15%) were non-Beijing genotype. Drug resistance rate of Beijing genotype strains was higher than that of non-Beijing genotype strains (32.14% vs 10.83%, P < 0.05). The resolution of VNTR-9 locus to Beijing genotype strains was 0.998 5 and the clustering rate was 12.15%. Conclusion Beijing genotype strains showed a high prevalence trend in Yichang area, drug resistance rate of Beijing genotype strains is higher. VNTR-9 genotyping method used to identify Beijing genotype strains of Mtb in this area can accurately reflect the molecular epidemiological characteristics of Mtb in Yichang.