Resistant Mycobacterium tuberculosis has become a serious threat to public health. In order to inhibit spreading and give effective treatment, it is of great importance, as early as possible, to detect drug-resistant bacteria. To evaluate the usefulness of the Line Probe Assay (INNO-LiPA^TM Rif.TB) for rapid detection of rifampicin resistance, we used 52 clinical isolates of M. tuberculosis from the national strain collection at the Swedish Institute for Infectious Disease Control and the drug susceptible reference strain H37Rv. By using BACTEC 460 methodology, 27 of these strains were determined as resistant to rifampicin and 26 as sensitive to rifampicin. Mutations known to give resistance to rifampicin were detected by LiPA in all 27 rifampicin-resistant strains. Among the 26 susceptible strains, 24 had the wild-type pattern in LiPA, while in two, mutations were seen. The LiPA correctly identified the M. tuberculosis complex in all samples. The high accuracy and simplicity of LiPA makes it a very promising method for the early identification of rifampicin resistance in M. tuberculosis.