Early detection of Mycobacterium tuberculosis (Mtb) in clinical specimens, its susceptibility to anti-TB drugs and disruption of infection transmission to new hosts are essential components for global tuberculosis (TB) control efforts. This study investigated major Mtb genotypes circulating in Kuwait and evaluated the performance of REBA MTB-MDR (REBA) test in comparison to GenoType MTBDRplus (gMTBDR+) assay for rapid detection of resistance of Mtb to isoniazid and rifampicin (MDR-TB). M. tuberculosis isolates (n = 256) originating predominantly from expatriate patients during a 6-month period were tested by spoligotyping and a dendrogram was created by UPGMA using MIRU-VNTRplus software. Phenotypic drug susceptibility testing (DST) was performed by MGIT 960 system. Genotypic DST for isoniazid and rifampicin was done by REBA and gMTBDR+ assays. Spoligotyping assigned 188 (73.4%) isolates to specific spoligotype international type (SIT) while 68 isolates exhibited orphan patterns. All major M. tuberculosis lineages were detected and EAI, CAS and Beijing families were predominant. Phylogenetic tree showed 131 patterns with 105 isolates exhibiting a unique pattern while 151 isolates clustered in 26 patterns. Fifteen isolates were resistant to one/more drugs. REBA and gMTBDR+ detected isoniazid resistance in 11/12 and 10/12 and rifampicin resistance in 4/5 and 4/5 resistant isolates, respectively. The diversity of SIT patterns are highly suggestive of infection of most expatriate patients with unique Mtb strains, likely acquired in their native countries before their arrival in Kuwait. Both, REBA and gMTBDR+ assays performed similarly for detection of resistance of Mtb to isoniazid and rifampicin for rapid detection of MDR-TB.