b Ethambutol (EMB) is an essential first-line drug for tuberculosis (TB) treatment. Nucleotide substitutions at embB codon 306 (embB306) have been proposed to be a potential marker for EMB resistance and a predictor of broad drug resistance in clinical Mycobacterium tuberculosis isolates. However, discordant findings about the association between embB306 mutations and EMB resistance were reported. Hebei Province is located in the Beijing-Tianjin-Hebei integration region in China; however, little information about the genetic diversity of the embB locus in this area is available. In this study, we sequenced the region surrounding embB306 (codons 207 to 445) in 62 ethambutol-resistant (EMB r ) isolates, 214 ethambutol-susceptible isolates resistant to other first-line drugs (EMB s isolates), and 100 pan-sensitive isolates. Our data indicated that none of the pan-sensitive isolates showed mutations at embB306 and 63 drug-resistant isolates harbored embB306 substitutions, with these substitutions being found in 56.5% (35/62) of EMB r isolates and 13.1% (28/214) of EMB s isolates. A significant association between the embB306 mutation and resistance to isoniazid, rifampin, EMB, and multiple drugs was observed, and the rate of mutation of embB306 increased with increasing numbers of first-line drugs to which the isolates were resistant. The embB306 mutation is not the sole causative factor for EMB resistance, and the poor sensitivity limits its utility as a marker for drug-resistant TB. However, it may be a potential marker for broad drug resistance, especially for multidrug resistance. The mycobacterial interspersed repetitive unit-variable-number tandem-repeat profiles may serve as markers for predicting the embB306 substitutions that may occur in drug-resistant M. tuberculosis isolates under antimicrobial selection pressure.T uberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis. According to estimates of the World Health Organization (WHO), in 2014 there were 9.6 million new TB cases and 1.5 million people died from this infectious disease (1). The widespread occurrence of drug-resistant (DR) TB (DR-TB), especially multidrug-resistant (MDR) TB (MDR-TB), has become a major global public health concern. In 2014, an estimated 3.3% of new TB cases and 20% of previously treated cases had MDR-TB and approximately 190,000 people died of MDR-TB worldwide. Treatment for MDR-TB is longer and requires more expensive and more toxic second-line drugs.Ethambutol (EMB) is an essential first-line drug for TB treatment and plays an important role in the chemotherapy of DR-TB (2). EMB acts against M. tuberculosis through inhibiting the membrane-associated arabinosyl transferases that are encoded by the embCAB operon (which included three contiguous genes, embC, embA, and embB) and that participate in the synthesis of arabinogalactan and lipoarabinomannan of the mycobacterial cell wall (3, 4). Mutations in the embCAB operon have been identified to confer resistance to EMB (3), with embB codon 306 (embB306) b...