ABSTRACT With Mycobacterium tuberculosis H37Rv as a control, the mutations of rpoB gene from 37 Mycobacterium tuberculosis clinical isolates (18 rifampin-resistant strains and 19 rifampin-susceptible strains) have been detected by polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) under standard condition (no glycerol in gel). For the strains in which mutations had not been detected under standard condition, PCR-SSCP analysis was performed again on condition that 8% glycerol was added to gel. In addition, the PCR products of rpoB genes of 30 strains (18 rifampin-resistant strains and 12 rifampin-susceptible strains) were detected by DNA sequencing. After twice analyses of PCR-SSCP, in 18 rifampin-resistant strains, the patterns of 17 strains were found to be abnormal; and in 19 rifampin-susceptible strains, the patterns of 16 strains were found to be normal. Compared with routine drug susceptibility test, the sensitivity of PCR-SSCP 94.4% is higher for rifampin resistance detection, and specificity of PCR -SSCP 84% seems lower. The results of sequence analysis were shown as: amomg 18 rifampin-resistant strains, only one has deletion in codons 513 and 514 of rpoB gene, which is a new report, the others have point mutation; among 12 rifampin- susceptible strains, 3 positive strains in SSCP occur gene mutations directly related to rifampin resistance. Then compared with DNA sequencing, the accuracy, sensitivity and specificity of PCR-SSCP are 96.7%, 95.2% and 100% respectively. Therefore, the sensitivity of mutation detection in SSCP could be improved by adding glycerol in gel. It is feasible and efficient to detect the mutation of rpoB gene in Mycobacterium tuberculosis by PCR-SSCP. This method is valuable to evaluate the rifampin resistance of Mycobacterium tuberculosis in clinical application for curing tuberculosis.