Molecular detection of bedaquiline resistant tuberculosis is challenging as only a small proportion of mutations in candidate bedaquiline resistance genes have been statistically associated with phenotypic resistance. We introduced two mutations,atpEIle66Val andRv0678Thr33Ala, in theMycobacterium tuberculosisH37Rv reference strain using homologous recombineering or recombination to investigate the phenotypic effect of these mutations. The genotype of the resulting strains was confirmed by Sanger- and whole genome sequencing, and bedaquiline susceptibility was assessed by minimal inhibitory concentration (MIC) assays. The impact of the mutations on protein stability and interactions was predicted using mutation Cutoff Scanning Matrix (mCSM) tools. TheatpEIle66Val mutation did not elevate the MIC above the critical concentration (MIC 0.25–0.5 µg/ml), while the MIC of theRv0678Thr33Ala mutant strains (> 1.0 µg/ml) classifies the strain as resistant, confirming clinical findings. In silico analyses confirmed that theatpEIle66Val mutation minimally disrupts the bedaquiline-ATP synthase interaction, while theRv0678Thr33Ala mutation substantially affects the DNA binding affinity of the MmpR transcriptional repressor. Based on a combination of wet-lab and computational methods, our results suggest that theRv0678Thr33Ala mutation confers resistance to BDQ, while theatpEIle66Val mutation does not, but definite proof can only be provided by complementation studies given the presence of secondary mutations.