Aims: The aim of this study was to investigate the features of rpoB gene mutations associated with Rifampin (RIF) resistance in Mycobacterium tuberculosis (M. tuberculosis) in eastern China. Methods and Results: The mutations of rpoB gene in 56 clinical isolates of M. tuberculosis resisted to one to four first-line drugs (rifampin, isonicotinyl hydrazide, ethambutol and streptomycin) were analysed by polymerase chain reaction single strand conformation polymorphism analysis (PCR-SSCP) and DNA sequencing. The results of PCR-SSCP showed 52 isolates were positive (existing rpoB mutation) including 47 isolates resisted to RIF. Subsequent results of DNA sequencing showed that 54 isolates had rpoB gene mutation including 49 isolates resisted to RIF. The most frequently mutated sites were at codons 526 (73.2%), 513 (10.7%) and 531 (3.5%). Conclusions: The rpoB codon 526 was the most frequently mutated site of RIF-resistant M. tuberculosis strains in eastern China and its frequency is significantly higher (P < 0.0001) compared with that in other areas of China and in other geographic regions worldwide. Significance and Impace of the Study: Our results reveal that geographic variation is responsible for rpoB mutations in M. tuberculosis and the resulting information will be helpful to improve a novel rapid molecular drug resistance screening approach for MDR TB.