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Ethambutol (EMB) is used as a part of drug regimens for treatment of tuberculosis (TB). Susceptibility ofMycobacterium tuberculosiscomplex (MTBC) isolates to EMB can be discerned by DNA sequencing to detect mutations in theembBgene associated with resistance. US Public Health Laboratories (PHL) primarily use growth-based drug susceptibility test (DST) methods to determine EMB resistance. The Centers for Disease Control and Prevention (CDC) provides a service for molecular detection of drug resistance (MDDR) by DNA sequencing and concurrent growth-based DST using agar proportion. PHL and CDC test results were compared for 211 MTBC samples submitted to CDC from September 2009 through February 2011. Concordance between growth-based DST results from PHL and CDC was 88.2%. A growth-based comparison of 39 samples, where anembBmutation associated with EMB resistance was detected, revealed a higher percentage of EMB resistance by CDC (84.6%) than by PHL (59.0%) which was significant (Pvalue = 0.002). Discordance between all growth-based test results from PHL and CDC was also significant (Pvalue = 0.003). Most discordance was linked to false susceptibility using the BACTEC™MGIT™960 (MGIT) growth-based system. Our analysis supports coalescing growth-based and molecular results for an informed interpretation of potential EMB resistance.