The nitroimidazole prodrugs delamanid and pretomanid are in advanced clinical development to treat tuberculosis (TB). These drugs comprise one of only two new classes of antitubercular agents to be approved for use within the last 50 years and are active against strains resistant to first-line drugs. They are administered as prodrugs, which undergo reductive activation by the deazaflavin (F420H2) dependent nitroreductase (Ddn). To ensure these drugs remain effective for as long as possible, it is important that we establish how resistance to these new drugs could develop after their introduction. Here, we reveal the role of Ddn in the energetics of M. tuberculosis through the reduction of menaquinone and showed its importance in the recovery from hypoxia. Analysis of ~15,000 sequenced M. tuberculosis genomes revealed ~1.5% of M. tuberculosis strains have non-synonymous mutations in the ddn gene. We tested the activity of more than 70 Ddn mutants with the prodrugs and quinones, including all point mutations found in the sequenced genomes. We identified mutations that can prevent pretomanid activation without significantly affecting its physiological role, thereby potentially conferring transmissible resistance. We confirmed that a transmissible M. tuberculosis isolate from the hypervirulent Beijing family already possesses one such mutation and is resistant to pretomanid, even though it has never been exposed to the drug. Notably, delamanid was still effective against this strain, which is consistent with our structural analysis that indicates delamanid and pretomanid bind to Ddn in different conformations. We suggest that SNPs identified in this work be monitored for informed pretomanid treatment and to slow the emergence of resistance.