Molecular methods detect genetic mutations associated with drug resistance. This study detected resistance-conferring mutations in gyrA/ gyrB for fluoroquinolones and rrs/ eis genes for second-line injectable drugs (SLIDs) among multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates in Kuwait. Fifty pansusceptible M. tuberculosis and 102 MDR-TB strains were tested. Phenotypic susceptibility testing was performed by MGIT 960 system using SIRE drug kit. GenoType MTBDR sl version 1 (gMTBDR slv1) and GenoType MTBDR sl version 2 (gMTBDR slv2) tests were used for mutation detection. Results were validated by PCR-sequencing of respective genes. Fingerprinting was performed by spoligotyping. No mutations were detected in pansusceptible isolates. gMTBDR slv1 detected gyrA mutations in 12 and rrs mutations in 8 MDR-TB isolates. gMTBDR sl2 additionally detected gyrB mutations in 2 and eis mutation in 1 isolate. Mutations in both gyrA/gyrB and rrs/eis were not detected. gMTBDR slv1 also detected ethambutol resistance-conferring embB mutations in 59 isolates. Although XDR-TB was not detected, frequency of resistance-conferring mutations for fluoroquinolones or SLIDs was significantly higher among isolates collected during 2013–2019 versus 2006–2012. Application of both tests is warranted for proper management of MDR-TB patients in Kuwait as gMTBDR slv2 detected resistance to fluoroquinolones and/or SLIDs in 3 additional isolates while gMTBDR slv1 additionally detected resistance to ethambutol in 58% of MDR-TB isolates.