embB306 mutations are potential markers for detecting ethambutol resistance in clinical Mycobacterium tuberculosis isolates. However, more recently, embB306 mutations have been found in ethambutol susceptible isolates and an association with broad drug resistance rather than ethambutol resistance has been reported. To further investigate this question, we analyzed the association between embB306 mutations and phenotypic ethambutol resistance among 197 isolates from a drug resistance survey performed in Karakalpakstan, Uzbekistan. 39 strains had an embB306 mutation, out of which seven were ethambutol susceptible, thus, displaying discrepant test results. After re-analysis, the seven isolates were tested ethambutol resistant. All of these strains had an increased ethambutol MIC, however, three strains showed no or weak growth on the critical concentration of 2 microg/ml on Löwenstein-Jensen. In three strains we confirmed the presence of heteroresistant mixed populations which might influence conventional ethambutol testing. Final concordance between molecular and phenotypic EMB testing was high with a sensitivity of 78% and a specificity of 100%. Our results confirm that embB306 mutations are useful markers for predicting ethambutol resistance. Discrepancies between molecular and phenotypic ethambutol resistance test results are most likely caused by problems with conventional susceptibility testing.