Drug-resistant tuberculosis is a serious health problem throughout the world. The aim of this study was to investigate the molecular epidemiology of rifampicin resistant Mycobacterium tuberculosis strains isolated in Manisa, Western Turkey. We evaluated the mutations in a 329 base pairs located in 481-589 codons of the rpoB gene by automated sequencing of 43 strains. In 43 rifampicin resistant strains, mutations were identified in 93% strains. No mutation was detected in the 3 resistant strains. The most common mutation (63%) was Ser531Leu. Overall, 15 different mutations and 13 alleles affecting 12 codons were identified. One new mutation (Ile572Leu), which was localized outside the RRDR was identified. The codon numbers of the most frequently encountered mutations were 531 (67%), 516 (9%), and 526 (5%). It was concluded that the results from this study can serve as a basis for monitoring molecular epidemiology of drug resistant M. tuberculosis strains isolated in our region.