Abstract: Objective　To investigate the molecular epidemiological characteristics of Mycobacterium tuberculosis in the southern region of Xinjiang, analyze the influencing factors of Mycobacterium tuberculosis strains clustering in the region, and provide a scientific basis for the prevention and treatment of tuberculosis in the southern region of Xinjiang. Methods Mycobacterium tuberculosis strains were randomly selected from the drug resistance monitoring sites in the southern region of Xinjiang Uygur Autonomous Region from January 2018 to December 2022 (except 2019). The DNA of the strains was extracted, and the strains were genotyped by the 24-locus VNTR method. The diversity and discriminatory power of loci were assessed, and the cluster rate and transmission differences of strains in Southern Xinjiang were analyzed. Results A total of 116 Mycobacterium tuberculosis strains were divided into 90 genotypes, including 76 unique genotypes, and the remaining strains formed 14 clusters of 2-7 strains each, totaling 40 strains, with a cluster formation rate of 34.48% and a transmission rate of 22.41%. The highest resolution index was observed at the QUB-11b (0.801) locus, with a discriminatory index of 0.801, while the lowest was at the MIRU2 locus, with a discriminatory index of 0. Beijing genotype accounted for 44.83% (52/116) of the MTB strains in southern Xinjiang. Statistical significance was found between strain clustering and resistance to isoniazid and rifampicin (χ2=6.798, P=0.009; χ2=15.544, P<0.001). Conclusions The genotypes of Mycobacterium tuberculosis in southern Xinjiang are highly polymorphic, but the Beijing genotype remains the predominant strain. The clustering rate of Mycobacterium tuberculosis in southern Xinjiang is higher, suggesting that the recent transmission of Mycobacterium tuberculosis in southern Xinjiang accounts for a certain proportion of the epidemic. In southern Xinjiang, Beijing genotype, rifampicin-resistant strains, and ethambutol-resistant strains are more likely to be clustered than non-Beijing genotype, rifampicin-resistant strains, and ethambutol-resistant strains.