Drug-resistant tuberculosis is a serious global health threat. Bedaquiline (BDQ) is a relatively new core drug, targeting the respiratory chain inMycobacterium tuberculosis(Mtb). While mutations in the BDQ target gene,atpE,are rare in clinical isolates, mutations in theRv0678gene, a transcriptional repressor regulating the efflux pump MmpS5-MmpL5,are increasingly observed, and have been linked to worse treatment outcomes. Nevertheless, underlying mechanisms of (cross)-resistance remain incompletely resolved. Our study aims to distinguish resistance associated variants from other polymorphisms, by assessing the in vitro onset of mutations under drug pressure, combined with their impact on minimum inhibitory concentrations (MICs) and on protein stability. For this purpose, isolates were exposed in vitro to sub-lethal concentrations of BDQ or clofazimine (CFZ). Selected colonies had BDQ- and CFZ-MICs determined on 7H10 and 7H11 agar. Sanger sequencing and additional Deeplex Myc-TB and whole genome sequencing (WGS) for a subset of isolates were used to search for mutations inRv0678, atpEandpepQ. In silico characterization of relevant mutations was performed using computational tools. We found that colonies that grew on BDQ medium had mutations inRv0678,atpEorpepQ, while CFZ-exposed isolates presented mutations inRv0678andpepQ, but none inatpE. Twenty-eightRv0678mutations had previously been described among in vitro selected mutants or in patients’ isolates, while 85 were new. Mutations were scattered across theRv0678gene without apparent hotspot. While mostRv0678mutations led to an increased BDQ- and/or CFZ-MIC, only a part of them surpassed the critical concentration (69.1% for BDQ and 87.9% for CFZ). Among the mutations leading to elevated MICs for BDQ and CFZ, we report a synonymous Val1Val mutation in theRv0678start codon. Finally,in silico characterization ofRv0678mutations suggests that especially the C46R mutant may renderRv0678less stable.