A combination of targeted molecular methods and phenotypic drug-susceptibility testing is the most widely used approach in clinic for drug resistance detection in Mycobacterium tuberculosis isolates. We report the delay in the introduction of an efficient anti-tuberculous drug regimen for a patient due to a M. tuberculosis strain displaying a high level of resistance to isoniazid, in the absence of the common mutations associated with isoniazid-resistance including katG mutations and inhA promoter mutations. Using whole genome sequencing, we identified in the isolate a large loss-of-function insertion (>1000pb) at the end of katG together with a -57C>T ahpC mutation; a resistance mechanism that would remain undetected by conventional molecular targeted approach. A retrospective search using publically available WGS data of more than 1200 isoniazid-resistant isolates and a similar sized control dataset of isoniazid-susceptible isolates revealed that most (22/31) isoniazid-resistant, KatG loss-of-function mutants had an associated rare ahpC promoter mutation. In contrast, only 7 out of 1411 isoniazid-susceptible strains carried a rare ahpC promoter mutation, including shared mutations with the 31 isoniazid-resistant, KatG loss-of-function mutants. Our study suggests that rare ahpC promoter mutations could be used as a proxy for investigating simultaneous KatG loss-of-function or missense mutations. We also conclude applying whole genome sequencing in routine diagnostic would improve drug susceptibility testing in M. tuberculosis clinical isolates and is an efficient tool for detecting resistance mechanisms undetected by conventional molecular methods.Copyright © 2020. Published by Elsevier B.V.