Objectives: Molecular assays to detect drug resistance in Mycobacterium tuberculosis are more rapid than standard drug susceptibility testing. To evaluate the efficacy of such assays in this setting, the GenoType® MTBDR plus assay (HAIN Lifescience) and multiplex allele-specific PCR assays were carried out. Methods: The GenoType® MTBDR plus assay was evaluated for the detection of rifampicin and isoniazid resistance in 223 M. tuberculosis isolates of known phenotypic drug sensitivity. The presence of KatG S315T and inhA C-15T mutations that confer isoniazid resistance was determined using multiplex allele-specific PCR assays. The relationship between isolate lineage and resistance determinant was investigated by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat analysis. Results: The GenoType® MTBDR plus assay detected multidrug-resistant, isoniazid-monoresistant and rifampicin-monoresistant isolates with sensitivities of 91.5%, 56.1% and 70%, respectively. Multiplex allele-specific PCR detected isoniazid resistance in 91.5% of the MDR isolates and 53.7% of the isoniazid-monoresistant isolates. The W-Beijing lineage was overrepresented in the MDR subgroup of strains (odds ratio, 3.29; 95% confidence interval, 1.76-6.16). Conclusions: A proportion of isoniazid resistance, particularly in isoniazid-monoresistant isolates of lineage X3, is due to resistance determinants other than KatG S315T and inhA C-15T. The fact that these isolates will be indicated as drug susceptible highlights the need for determining local patterns of resistance mutations to provide users with information regarding the capabilities of rapid genotypic assays.