Mutations of the 81-bp-core region of rpoB gene associated with rifampicin resistance were studied in 169 clinical strains of Mycobacterium tuberculosis isolated from Spain between 1996 and 2001. The analysis identified 16 single base changes, one deletion, two insertions and five multiple mutations (with two or three codons implicated). Eight strains had no mutations, although they were resistant by the proportion method. The five single nucleotide changes with two or three codons implicated and one insertion have not been previously reported. The analysis of the RFLP-IS6110 pattern showed a great heterogeneity (159 individual patterns and four patterns with two or three strains) that suggests no geographical or temporal relations between the studied strains. The concordance rate of InnoLipa Rif-Tb probe with sequencing and phenotypic methods was 94.6%. The results indicate that the InnoLipa probe assay may be useful for rapid screening of the most frequent mutations found in rifampicin-resistant M. tuberculosis strains.