Drug resistance in tuberculosis (TB) has become a major public health threat, particularly when the disease cannot be 100% controlled by BCG vaccination. In Thailand, resistance to rifampicin, a major component of multidrug regimens of treatment, is the common cause of tuberculosis recurrence. The mechanism of rifampicin resistance involves alterations of the RNA polymerase subunit beta (rpo B) gene. Mutations in rpo B gene were often found to cluster within a region of 23 amino acids starting from amino acid residue 511 to residue 533. Direct PCR sequencing was utilized to compare base changes in rpo B gene in three rifampicin resistant phenotypes of M. tuberculosis isolated from Thai patients. The sequences showed one base substitution at codon 531 resulting in an amino acid change from serine (TCG) to leucine (TTG) in a multidrug resistant isolate compared to that of a sensitive isolate, whereas a point mutation at codon 516 causing a change from aspartic acid (GAC) to tyrosine (TAC) was detected in a multidrug resistant isolate from a HIV positive patient. In an isolate resistant only to rifampicin a double mutation at codon 531 changing serine (TCG) to phenylalanine (TTT) was found. No mutations were observed in the same region in streptomycin, ethambutol or isoniazid resistant isolates. This finding reports two new types of mutation (GAC to TAC at codon 516 and TCG to TTT at codon 531) and confirms a direct correlation between rpo B gene alteration and rifampicin resistant phenotype in M. tuberculosis.