Tuberculosis (TB) has been a threat to world health for decades. Multidrug-resistant TB (MDR-TB) is adding to the burden of disease and hindering the development of countries. This study aimed to identify the drug-resistant rate in patients with recurrent pulmonary TB and detect rifampicin resistance mutations in the rpoB gene in Can Tho, Vietnam. In this descriptive cross-sectional study, clinicians diagnosed the patients with recurrent pulmonary TB and were hospitalized. Smear-positive sputum specimens (n=246) were collected to determine the rate of drug resistance. To identify mutations in the rpoB gene which are likely to be associated with rifampicin resistance in MDR-TB, we sequenced 40 isolates of Mycobacterium tuberculosis collected between 2012 and 2014; mainly MDR-TB (n=40; 95.2%) and rifampicin-sensitive TB (n=2; 4.8%). The rate of drug-resistant pulmonary tuberculosis was 63.8%, and the rate of MDR-TB accounted for 23.2%. The mutations in rpoB were predominantly in codons 531 (27.5%) and 523 (17.5%), with rare occurrences of S522A (2.5%) and A532P (2.5%). Noticeably, there was a substitution mutation in codon 532, and a mutant strain of tuberculosis at seven codons in the rpoB gene had been detected. In conclusion, this study provided drug-resistant characteristics in patients with recurrent pulmonary TB and mutations of MDR-TB in Can Tho, Vietnam. The results show a mutation in codon 532 of the rpoB gene and a mutant strain of tuberculosis at all seven codons. These are remarkable and promising results for further studies and clinical applications.