Streptomycin (SM) displays antimicrobial activity via inhibition of protein synthesis in bacteria and it is one of the major antituberculous drugs used in chemotherapy of tuberculosis (TB). The aim of this study was to investigate the presence of mutations in rpsL and rrs gene regions responsible for SM resistance using DNA sequencing system in SM resistant Mycobacterium tuberculosis complex (MTC) isolates detected by a phenotypic drug susceptibility test. Ten SM resistant and 10 SM sensitive MTC strains isolated between 2003-2007, in Mersin University Medical Faculty Department of Medical Microbiology Laboratory, were included to this study. The identification of the strains and their susceptibility to primary antituberculous drugs were performed by BACTEC 460 TB system (Becton Dickinson, USA). The SM susceptibility was retested by agar proportion method at low (2 microg/ml) and high (10 microg/ml) concentrations of SM. For genotypic analysis, isolated DNA samples were amplified with polymerase chain reaction (PCR) using primers specific for rpsL and rrs gene regions associated with SM resistance. PCR products were analyzed for the presence of nucleotide changes by silver stained DNA sequence analysis (Silver Sequence DNA Sequencing System, Promega) and the data were compared to sequences encoded as L08011 for rpsL gene region and X52917 for rrs gene region in GeneBank database. Of the 10 SM resistant isolates 8 were resistant to SM at both low and high drug concentrations (2 microg/ml and 10 microg/ml) while two were resistant to SM at low drug concentration (2 microg/ml) using agar proportion method. A point mutation as AAG-->AGG (Lys-->Arg) was detected at codon 43 of rpsL gene region in eight isolates resistant to SM at both low and high drug concentrations. Nucleotide changes associated with mutation were not detected in 10 sensitive isolates and two low drug concentration resistant isolates. It was concluded that molecular analysis of MTC isolates might aid to the success of TB treatment and larger scale nationwide studies should be conducted to enlighten the molecular basis of drug resistance in MTC isolates.