Mutations in the rpoB gene associated with rifampicin (RMP) resistance were studied in 47 RMP-resistant and 147 RMP-susceptible clinical strains of Mycobacterium tuberculosis from Morocco using probe-based assay and DNA sequencing. RMP-resistant mutations were identified in 85% of RMP-resistant isolates. No mutations were observed among the 147 RMP-susceptible strains. Sequence analysis identified 10 alleles, including two deletions not previously reported. Nucleotide changes at codons 531, 526 and 516 were the most prominent, accounting for 74.4% of our RMP-resistant strains. These results demonstrate that resistance genotyping at these codons would be a good marker for the rapid detection of RMP resistance.