Background & objectives: Frequency of resistance-conferring mutations vary among isoniazid- and ethambutol-resistant Mycobacterium tuberculosis isolates obtained from patients of various ethnic groups. This study was aimed to determine the occurrence of specific rpoB mutations in rifampicin-resistant M. tuberculosis isolates from tuberculosis patients of various ethnic groups in Kuwait. Methods: Rifampicin-resistant M. tuberculosis isolates (n=119) from South Asian (n=55), Southeast Asian (n=23), Middle Eastern (n=39) and other (n=2) patients and 107 rifampicin-susceptible isolates were tested. Mutations in rpoB were detected by DNA sequencing. Polymorphisms at katG463 and gyrA95 were detected by PCR-RFLP for genetic group assignment. Results: None of rifampicin-susceptible but 116 of 119 rifampicin-resistant isolates showed rpoB mutation(s). Mutations among isolates from South Asian patients were distributed at rpoB516 (20%), rpoB526 (24%) and rpoB531 (27%) while 78 and 51 per cent of isolates from Southeast Asian and Middle Eastern patients, respectively, contained a mutated rpoB531. All isolates with rpoB N-terminal and cluster II mutations were obtained from Middle Eastern and South Asian patients. Most isolates from South Asian (84%) and Southeast Asian (70%) patients belonged to genetic group I while nearly all remaining isolates belonged to genetic group II. Isolates from Middle Eastern patients were distributed among genetic group I (46%), genetic group II (33%) and genetic group III (21%). Interpretation & conclusions: The occurrence of specific rpoB mutations varied considerably in rifampicin-resistant M. tuberculosis isolates obtained from patients of different ethnic groups within the same country. The present data have important implications for designing region-specific rapid methods for detecting majority of rifampicin-resistant strains.