Introduction: Tuberculosis (TB) still remains a public health problem worldwide and the emergence of drug resistant TB (DR-TB) has worsened the situation as it is difficult and expensive to treat. The characterization of the genetic mutations underlying streptomycin resistance may be helpful in developing rapid detection methods which may guide clinicians in making therapeutic decisions. The aim of this study is to detect mutations causing streptomycin (STR) resistance in Mycobacterium tuberculosis isolates from Sabah. Methods: Susceptibility testing was carried out in MGIT system for 42 Mycobacterium tuberculosis clinical isolates. The drug resistant isolates were subject to whole genome sequencing and in-silico analysis was performed to detect the mutations in the sequence of the rpsL gene known to confer resistance to anti-tuberculous drugs. Results: Of the 42 positive isolates, 27 (64.3%) are shown to be susceptible towards first line drugs (FLDs) while 15 (35.7%) isolates were mono- and multiple resistant to the FLDs. Our findings reveal that the isolate 145 possess mutations at codon 43 within rpsL gene with amino acid change A to G (K43R). Conclusion: Findings from this study enable us to expand our knowledge of mutations causing drug resistance in Mycobacterium tuberculosis and the point mutations, which can be used as the potential marker for detection of drug resistant isolates.