A total of 196 rifampicin (RMP) resistant Mycobacterium tuberculosis isolates were searched for mutations in the cluster I region of the rpoB gene. Seventeen different types of mutations were observed in 184 (93.9%) isolates. The remaining isolates searched also had no mutations in the N-terminal region of this gene. Mutations were most frequently detected at single codons Ser531 (47.4%), His526 (20.9%), and Asp516 (20.4%). Our data indicate that genotypic drug susceptibility assays based on detection of mutations at the specific codons of the rpoB gene should be effective for predicting resistance to RMP and rifabutin and multidrug resistance in Lithuania.