The aim of this study was to characterize gene mutations in (MTB) and investigate the factors associated with mutations and the relation between mutations and tuberculosis (TB) transmission. A total of 245 MTB clinical isolates from patients with TB in six provinces and two municipalities in China were characterized based on gene mutations through DNA sequencing of and genes, phenotyping via standard drug susceptibility testing, and genotypic profiling by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing. Approximately 36.4% of the rifampin-resistant isolates harbored nonsynonymous mutations in the gene. Twenty-nine nonsynonymous single mutations and three double mutations were identified. The mutations at locus 483 (11.3%) were predominant, and the mutations at V483G, W484G, I491V, L516P, L566R, N698K, and A788E accounted for 54.5% of the total detected mutations. Fifteen new mutations in the gene were identified. Rifampin resistance and mutations at locus 531 were significantly associated with mutations. MIRU-VNTR genotype results indicated that 18.4% of the studied isolates were clustered, and the mutations were not significantly associated with MIRU-VNTR clusters. A large proportion of mutation was observed in the rifampicin-resistant MTB isolates. However, the findings of this study do not support the association of mutation with compensated transmissibility.