This study investigated mutations in the rpoB gene of rifampin-resistant isolates obtained from patients living in Eastern Poland. A total of 37 phenotypically and/or genotypically confirmed M. tuberculosis rifampin-resistant clinical isolates were included in this study. The strains were selected from symptomatic patients with a diagnosis of pulmonary and extrapulmonary tuberculosis. A line probe assay kit (INNO-LiPA rif Tb) was used for any specific mutational pattern of rpoB gene. Our data support the common notion that rifampin resistance genotypes with mutation at a critical codon, i.e. the one encoding Ser-531, is frequent in M. tuberculosis populations regardless of geographic origin. Our findings also suggest that in a geographic area such as Eastern Poland less common mutations of the rpoB gene occur more frequently. The frequency of substitution at codon 526 (His-Asp) was found to be high in Lublin. This study indicates that mutations associated with nucleotide replacements in codons 526 (His-Asp) and 531 (Ser-Leu) were associated with a high percentage of RMP resistance, whereas mutations in codons 516 (Asp-Val) and 526 (His-Tyr) were observed in a low percentage of RMP-resistance.