The mutation of the rpsL gene in Mycobacterium tuberculosis (M.TB) is related to Streptomycin resistance. In this experiment, 77 TB strains obtained in hospital were selected, the routine drug susceptibility test was done by using BACIEC460 system, meanwhile the TB-rspL gene was amplicated by PCR, and analyzed by SSCP, RFLP and sequence analysis. There are 20 strains sensitive to SM and 57 strains resistant to it in routine drug susceptibility test. The SSCP result displayed that 34 strains were rspL gene wild type and 43 strains were mutant type. Compared with routine drug susceptibility test, the specificity was 95% and the positive anticipate value was 98%. RFLP cut by MobII displayed that 81.4% of mutation happened in codon 43 of TB-rspL. Sequence analysis displayed that there was single base mutation in codon 43(AAG-->AGG). In conclusion, Drug resistance of TB to SM is related to rspL gene mutation and the mutation in codon 43 is the most common cause.