Abstract: Objective To investigate the molecular characteristics of clinical multi-drug resistant Mycobacterium (M.) tuberculosis (MDR-TB) isolates to three second line drugs (capreomycin, kanamycin andofloxacin) and the association with the Spoligotyping genotypes of M. tuberculosis in China. Methods A total of 128 MDR-TB were selected for Spoligotyping, then PCR and sequencing were conducted for the five selected genes (tlyA, eis, rrs, gyrA and gyrB) associated with capreomycin, kanamycin andofloxacin resistances. Results rrs-A1401G mutation occurred in 60.0% of the capreomycin resistant isolates (6/10) and 0.8% of the capreomycin sensitive isolates (1/118). For the kanamycin resistant isolates 84.6% had rrs or eis promote region mutations (11/13), in which 53.8% (7/13) had rrs-A1401G mutation, 23.1% (3/13) had eis-C(-10) A mutation, 7.7% (1/13) had eis-G(-14)T mutation, while 3.5% of the kanamycin sensitive isolates (4/115)had eis-C(-12)T mutation. For of loxacin resistant isolates, 83.3% (53/60) had gyrA mutation and 5% (3/60) had gryB mutation, the most frequent mutation in gryA gene was at the codon 94(37/60, 61.7%) and followed by codon 90(12/60, 20.0%). For all the selected isolates, 84.4% (108/128) were identified as Beijing genotype isolates, and no relationship was found between Spoligotyping genotypes and drug resistance phenotypes. The sensitivity and specificity for sequencing to identify the susceptibility to capreomycin, kanamycin andofloxacin were 60.0% and 99.4%, 84.6% and 99.4%, 90.0% and 100.0%, respectively. Conclusion The most frequent occurred mutation loci in the selected genes were rrs-1401, eis-10, and gyrA-94, -90, -91, which can be used as evidence for the rapid detection of drug susceptibility of M. tuberculosis.