ABSTRACT Ethambutol (EMB) resistance can evolve through a multistep process, and mutations in the ubiA ( Rv3806c ) gene appear to be responsible for high-level EMB resistance in Mycobacterium tuberculosis . We evaluated the prevalence of ubiA and embB ( Rv3795 ) mutations in EMB-resistant strains originating from Africa and South Korea. No differences in embB mutation frequencies were observed between strains from both origins. However, ubiA mutations were present in 45.5% ± 6.5% of the African EMB-resistant isolates but in only 9.5% ± 1.5% of the South Korean EMB-resistant isolates. The ubiA mutations associated with EMB resistance were localized to regions encoding the transmembrane domains of the protein, whereas the embB mutations were localized to regions encoding the extramembrane domains. Larger studies are needed to investigate the causes of increased ubiA mutations as a pathway to high-level EMB resistance in African countries, such as extended EMB usage during tuberculosis treatment.