Objective　To explore the molecular epidemiological characteristics of local Mycobacterium tuberculosis in Chongqing, China using genotyping methods and establish a suitable genotyping system tuberculosis genotyping in the region. Methods A total of 268 isolates collected from the sputum of tuberculosis patients at two national drug resistance monitoring sites in Chongqing from 2019 to 2021. The DNA of these isolates was extracted, and genotyping of the isolates was performed using VTNR 24 locus typing method. Epidemiological investigations were conducted on clustered isolates, and the clustering rates of isolates from the two surveillance sites were compared to analyze their transmission differences. Results In the two regions, the Hunter-Gaston discriminatory index (HGI) in Fengjie County ranged from 0 to 0.85, with 10 loci having an HGI above 0.6, 11 loci with an HGI between 0.3 and 0.6, and 3 loci with an HGI below 0.3; the highest resolution site was MIRU31, while the lowest resolution site was MIRU24. The HGI index in Fengdu County ranged from 0 to 0.81, with 12 loci having an HGI above 0.6, 9 loci with an HGI between 0.3 and 0.6, and 3 loci with an HGI below 0.3; the highest resolution site was MIRU26, while the lowest resolution site was MIRU23. In the clustering analysis, among the 140 isolates from Fengdu County, 10 samples clustered into 4 clusters, with the largest cluster consisting of 4 samples, resulting in a clustering rate of 4.1%. Among the 128 isolates in Fengjie County, 10 samples clustered into 5 clusters, with a clustering rate of 4.2%. Conclusion In Fengdu County and Fengjie County of Chongqing, the main population of the disease show independent genotypes, indicating a low recent transmission rate. The incidence of patients is mainly endogenous recurrence, that is, latent infection develops into active tuberculosis. While conducting standardized management of tuberculosis patients, prevention and control agencies should also increase the screening of latent infection at two monitoring points and carry out preventive treatment of latent infection, which is the key to reduce the incidence of tuberculosis in the two places.