Study carried out on 54 rifampicin-resistant M. tuberculosis strains and 20 susceptible strains that were collected in the North, Central and South of Vietnam in 528bp region of rpoB gene. All 20 susceptible strains had no mutations in the rpoB gene. 52/54 RIF-resistant strains showed mutations in the rpoB gene (96.3 percent). Sensitivity, specificity, negative predictive value, positive predictive value of rpoB gene sequencing method was 96.3 percent, 100 percent, 90.9 percent and 100 percent for detecting rifampicin resistance, respectively. Overall concordance of the rpoB gene sequencing technique with conventional drug susceptibility testing was very high (kappa value was 0.934) for detecting rifampicin resistance. rpoB gene sequencing technique can be applied in screening and diagnosis of rifampicin-resistant and also multi-drug resistant M. tuberculosis for high-risk patients.