The aim of the present study was to evaluate a new improved multiplex polymerase chain reaction (PCR) hybridisation assay to detect multidrug-resistant tuberculosis. The assay, developed to detect rifampin (rpoB) and isoniazid (katG) gene mutations causing Mycobacterium tuberculosis resistance, was recently extended to include inhA gene mutations that code for low-level isoniazid resistance. Interpretable results were obtained in 115 isolates and in all smear-positive clinical specimens. Rifampin resistance was correctly identified in all specimens and in 20 of 21 (95%) multidrug-resistant isolates compared to BACTEC 460TB. Isoniazid resistance correlated in 18 of 22 (82%) specimens, in 31 of 31 (100%) high-level and 24 of 28 (86%) low-level isoniazid-resistant isolates. The assay was rapid, easy to perform and directly applicable in smear-positive specimens. We predict that the assay may be a useful tool to combat and prevent new cases of multi- and extensively drug-resistant tuberculosis.