Ethambutol (EMB) is the first-line antituberculosis drug and a potential supplementary agent for a treatment regimen of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). It has long been known that mutations in embCAB operon, encoding EMB target, arabinosyltransferase, confer resistance to EMB. Recently, ubiA was additionally reported to be specifically associated with high-level EMB resistance in Mycobacterium tuberculosis. However, such information on ubiA is very limited. This study aimed to investigate correlations between mutations in ubiA and phenotypic EMB resistance among EMB-resistant (EMB^R) M. tuberculosis Thai clinical isolates. Minimum inhibitory concentration (MIC) level of EMB and ubiA sequences were determined and analyzed. Of 68 EMB^R-MDR isolates, 8.9% harbored mutations in ubiA. However, 10.0% and 46.6% of EMB-sensitive (EMB^S)-MDR and pan-susceptible isolates also had ubiA mutations detected, respectively. Most nonsynonymous mutations, L31P, A35S, and V55M were only found in the EMB^R-MDR isolates except E149D which was also found in EMB^S-MDR and pan-susceptible isolates. A further phylogenetic analysis based on spoligotyping and IS6110-RFLP illustrated that E149D was in fact associated to EAI-families rather than EMB resistance. By excluding synonymous mutations and the E149D, we found a high correlation between ubiA mutations and high-level of EMB resistance with 100.0% specificity. In conclusion, despite its rare occurrence, mutations in ubiA can potentially be a marker for a detection of high level of EMB resistance at least in the MDR M. tuberculosis background.