Background: The dual challenges to tuberculosis (TB) control of HIV infection and multi-drug resistance (MDR) are particularly pressing in South Africa. Conventional methods for detecting M. tuberculosis drug resistance take weeks to months to produce results. Rapid molecular testing for drug resistance is available but has not been implemented in high TB burden settings. Methods: We assessed the performance and feasibility of implementation of a commercially available molecular line-probe assay for rapid detection of rifampicin and isoniazid resistance directly on 536 consecutive smear-positive sputum specimens from patients at increased risk of MDR-TB in a busy routine diagnostic laboratory in Cape Town, South Africa. Results were compared with conventional liquid culture and drug susceptibility testing (DST) on solid medium. Results: Overall, 97% of smear-positive specimens gave interpretable results within 1-2 days using the molecular assay. Sensitivity, specificity, positive and negative predictive values were 98.9%, 99.4%, 97.9% and 99.7% for detection of rifampicin resistance; 94.2%, 99.7%, 99.1% and 97.9% for detection of isoniazid resistance; and 98.8%, 100%, 100% and 99.7% for detection of multidrug-resistance compared with conventional results. The assay also performed well on specimens that were contaminated on conventional culture and on smear-negative, culture-positive specimens. Conclusions: This molecular assay is a highly accurate screening tool for MDR-TB that achieves a substantial reduction in diagnostic delay. With overall performance characteristics that are superior to conventional culture and DST and the possibility for high throughput with substantial cost savings, molecular testing has the potential to revolutionize MDR TB diagnosis.