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The length and complexity of tuberculosis (TB) therapy, as well as the propensity ofMycobacterium tuberculosisto develop drug resistance, are major barriers to global TB control efforts.M. tuberculosisis known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a novel mechanism of genetically encoded but rapidly reversible drug-tolerance inM. tuberculosiscaused by transient frameshift mutations in a homopolymeric tract (HT) of seven cytosines (7C) in theglpKgene. Inactivating frameshift mutations associated with the 7C HT inglpKproduce small colonies that exhibit heritable multi-drug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the sameglpKHT region. These reversible frameshift mutations in the 7C HT ofM. tuberculosis glpKoccur in clinical isolates, accumulate inM. tuberculosisinfected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction ofdosRandsigHand repression ofkstRregulons, similar to that observed in otherin vitromodels ofM. tuberculosistolerance. These results suggest that GlpK phase variation may contribute to drug-tolerance, treatment failure and relapse in human TB. Drugs effective against phase-variantM. tuberculosismay hasten TB treatment and improve cure rates.SIGNIFICANCEThe ability ofM. tuberculosisto survive during prolonged treatment has been attributed to either transient stress responses or fixed heritable drug-resistance producing mutations. We show that phase-variation in theM. tuberculosis glpKgene represents a third type of resistance mechanism. The ability of theseglpKmutants to grow slowly and then rapidly revert suggests that these transiently-heritable changes may also explain how a hidden population of drug-tolerant bacteria develops during TB treatment. As a genetically trackable cause of drug-tolerance,M. tuberculosis glpKmutants provides a unique opportunity to study these phenomena at a cellular and mechanistic level. These mutants could also be used for developing drugs that target tolerant populations, leading to more rapid and effective TB treatments.