Objective To investigate the prevalence of mutation in the locus 306 of embB gene in multi-drug resistant (MDR) Mycobacterium tuberculosis (TB) and evaluate the prospects for using it as a molecular marker to detect MDR-TB.Methods The 291 strains enrolled in this study were from the reference laboratory of Shanghai municipal centers for disease control and prevention, all of which had been tested for drug susceptibility.Mutation in embB 306 was screened both by amplification refractory mutation system (ARMS) and DNA sequencing.The mutation frequencies of embB 306 in the sample groups varied in drug resistance were statistically analyzed.Results 38(51.4% ) of the 74 MDR-TB were embB 306-mutant (X2 =93.8,P＜0.01).Of the 24 TB resistant to at least two drugs but not MDR, 9(37.5% ) were embB 306 mutant (X2 =60.1 ,P＜0.01 ).But only two(4.9% ) embB 306-mutant strains were found in 41 strains resistant to only one drug (X2 =6.8,P=0.0093).None embB 306-mutant strains were found in 152 pansensitive strains.The specificity of using embB 306 as a molecular marker for detecting multi-drug resistant TB was 94.9% (206/217).Conclusions As a molecular marker for screening drug resistant TB,especially MDR-TB, the gene locus embB 306 shows a relatively high sensitivity and specificity, promising a sound future for its application in clinics to realize fast screening of patients infected with MDR-TB and to provide evidence for appropriate medication.