The objective of this study was for the elucidation of the characteristics of the rpoB gene mutation in rifampicin (RIF)-resistant Mycobacterium tuberculosis strains isolated in China. The rifampicin resistance determination regions (RRDR) of the rpoB genes of 242 M. tuberculosis strains were sequenced, including 193 RIF-resistant, 46 RIF-sensitive clinical isolates and three manually induced RIF-resistant tuberculosis strains. Mutations in the 81 bp RRDR of the rpoB gene were identified in 89.6% (173/193) of RIF-resistant clinical isolates. No mutation was observed in RIF-sensitive strains. Ser531Leu mutations accounted for 46.1% (89/193) of RIF-resistant strains; the mutation frequency of 526-His was 17.6% (34/193) in RIF-resistant strains. Furthermore, a combination of 2-3 single-point mutations was observed in 24 RIF-resistant strains (12.4%; 24/193). Mutations in three manually induced RIF-resistant tuberculosis strains were located at codons 531 and 526, respectively. The results indicate that about 90% of rifampicin-resistant M. tuberculosis strains isolated in China had rpoB mutations;531-Ser and 526-His were the most common positions substituted; high-level rifampicin-resistant strains had a higher frequency of 531-Ser mutations than low-level rifampicin-resistant strains. Sequencing analysis of the 81 bp fragment in the rpoB gene is useful in predicting the rifampicin-resistant phenotype.