Introduction: Rifampicin is an important antibiotics which was chosen to treat tuberculosis. However, anti-TB activity of rifampicin is now being reduced by the emergence of drug resistance of Mycobacterium tuberculosis strains. Objectives: To establish the method for rapid detection of rifampicin resistance mutations in Mycobacterium tuberculosis. Method: Bacteria culture was used to assess antibiotic resistance. To detect TB resistant to rifampicin, we used multiplex allele specific polymerase chain reaction assay (MAS-PCR) and compared the results with DNA sequencing. Results: 15 strains of TB were cultured successfully, among those, three strains were multidrug-resistant. The results showed that point mutation in the specific position rpoB516, rpoB526 and rpoB531 was detected through MAS-PCR assay within a day. The results of MAS-PCR were confirmed by rpoB gene sequencing. Compared with sequencing and bacterial culture techniques, the MAS-PCR was performed in a shorter time (1 day compared with 3 and 30 days).