Ethambutol (EMB) is an essential first-line drug for tuberculosis (TB) treatment. Nucleotide substitutions atembBcodon 306 have been proposed as a potential marker for EMB resistance and a predictor for broad drug resistance in clinicalMycobacterium tuberculosis(MTB) isolates. However, discordant findings about the association betweenembB306 mutation and EMB resistance were reported. The Hebei province is located in the Beijing-Tianjin-Hebei integration region; however, little information about the genetic diversity ofembBlocus is available in this area. In this study, we sequenced the region surroundingembB306 codon (codon 207-445) in 62 ethambutol-resistant (EMBr) isolates, 214 ethambutol-susceptible isolates but with resistance to other first-line drugs (EMBs) and 100 pan sensitive isolates. Our data indicated that none of pan sensitive isolates showed mutations atembB306 and 63 drug-resistant isolates harboredembB306 substitutions, with 56.5% (35/62) in EMBrisolates and 13.1% (28/214) in EMBsisolates. Significant association was observed between theembB306 mutation and resistance to INH, RIF, EMB and MDR, and the mutation rates ofembB306 increased with increasing numbers of resistance to first-line drugs. TheembB306 mutation is not the sole causative factor for EMB resistance and the poor sensitivity limits its utility as a marker for DR-TB. However, it may be a potential marker for broad resistance, especially for MDR. The MIRU-VNTR profiles may serve as a potential marker for predicting the possibleembB306 substitutions that may occur in DR-TB isolates under antimicrobial selection pressure.