Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the leading causes of death among the curable infectious diseases (WHO/IUATLD, 2004) despite the fact that the available short-course therapy proved to be cost effective. The emergency of multidrug-resistant TB (MDR-TB) caused by M. tuberculosis strains resistant to at least isoniazid (INH) and rifampicin (RMP) has worsened the situation and complicated the world’s efforts to control the disease. Conventional methods for detecting M. tuberculosis drug resistance take several weeks to months to obtain results. In this study we evaluated the sensitivity and specificity of a commercially available molecular line-probe assay GenoType® MTBDRplus for rapid detection of RMP and INH resistance directly on smear-positive sputum specimens preserved in 50% ethanol. A total of 321 smear-positive sputum specimens from repeat treatment TB cases obtained during the routine drug susceptibility survey conducted in Tanzania were evaluated. Susceptibility testing was performed on 205 isolates at the SRL, in Antwerp and on 285 isolates at the NRL in Tanzania.