Background: The global rate of antimicrobial resistance is rising for M. tuberculosis, the leading causative agent of tuberculosis infections. Gold standard phenotypic methods for drug susceptibility testing (DST) are slow and labor-intensive, but rapid DST is possible by using molecular predictors of resistance. Molecular DST for first-line anti-tuberculosis drugs ethambutol and pyrazinamide detects mutations in embB and pncA, respectively. However, discordance between phenotypic and molecular methods is described for both drugs. Aims: This study sought to (1) evaluate and compare ethambutol resistance using phenotypic and molecular DST on M. tuberculosis isolates containing embB406 mutations, and (2) investigate pyrazinamide resistance-associated mutations in isolates lacking pncA mutations. Methods: (1) Phenotypic DST for ethambutol was performed on the BACTEC™ MGIT™ 960 in duplicate by proportion method (2, 3, 4, and critical concentration 5 μg/mL). Whole genome sequencing was analyzed for drug resistance predictions, phylogenomics, and single nucleotide polymorphism analysis. (2) An M. tuberculosis outbreak cluster containing sensitive and resistant isolates lacking pncA mutations and seven closely related isolates were used to investigate mutations associated with the emergence of pyrazinamide resistance (i.e., critical concentration 100 ug/mL) using genomics and RNA sequencing-based transcriptomics. Results: (1) Sequencing revealed two embB406 mutation subgroups (Gly406Asp, Gly406Ala) in 16 isolates predicted to be ethambutol-resistant. However, 12 of these isolates were phenotypically sensitive at 5 µg/mL but resistant at 2-4 µg/mL. A novel frameshift mutation in regulator embR (gln258fs) was found in nine isolates with low-level resistance (2-4 µg/mL). (2) Pyrazinamide-resistant isolates contained resistant subpopulations with pncA mutations. Transcriptomics revealed differential expression of pncB2, genes involved in virulence factor synthesis (ppsC, ppsD, mtbC, mtbD), and probable ATP transporter Rv1686c/Rv1687c between sensitive and resistant isolates. Conclusions: (1) Mutations in embB406 are low-confidence and may be associated with low-level ethambutol resistance undetectable at the current critical concentration for ethambutol DST. Novel mutations outside embB are predicted to exacerbate variability in ethambutol susceptibility within isolates harbouring identical embB406 mutations. (2) The discordance observed between the molecular and phenotypic DST for pyrazinamide suggests that there might be additional genes or alternative mechanisms, apart from pncA, that could potentially play a role in pyrazinamide resistance.