OBJECTIVE To develop a pyrosequencing approach to rapid detection of rifampin resistance in Mycobacterium tuberculosis based on characterization of all possible mutations in the 81 bp core region. METHODS Two pyrosequencing sequencing primers and 1 pair of PCR primers were chosen for pyrosequencing analysis. The sensitivity of the pyrosequencing approach was determined by assaying PCR products generated from 10-fold serial dilutions of the DNA from Mycobacterium tuberculosis H(37) Rv strains. The efficacy of the pyrosequencing approach was evaluated by analyzing clinical Mycobacterium tuberculosis isolates with known antibiotic phenotypes. RESULTS Rifampin resistance could be determined within 2 hours after PCR amplification and single-stranded template preparation by using only two pyrosequencing reactions. About 50 fg DNA/reaction was required in order to get sufficient PCR product for producing a long, clear and accurate pyrosequencing pattern. A total of 41 Mycobacterium tuberculosis isolates were tested and the results were concordant with those based on drug susceptibility testing and conventional DNA sequencing. CONCLUSION The pyrosequencing technique is simple to perform and can be used as a rapid, high-throughput and efficient method for detecting rifampin resistance in Mycobacterium tuberculosis.