Abstract Mycobacteria populations can undergo mutations in their DNA sequence during replication, which if not repaired would be transferred to future generations. Earlier studies have tackled the estimation of mutation rate in mycobacteria at fixed concentrations. However, in this study, in vitro spontaneous mutations in Mycobacterium smegmatis ( Msm ) mc 2 155 ( Msm ) that confers resistance to some of the most important antitubercular drugs; isoniazid (INH r ), rifampicin (RIF r ), kanamycin (KAN r ) and streptomycin (STR r ) were first determined at several highly lethal concentrations, a few of which have not been previously investigated, in a fluctuation assay. Thereafter, mutation rate was estimated using the most commonly adopted Po method, and estimates were then compared concurrently with the Lea‐Coulson method of the median and Ma‐Sandri‐Sarkar Maximum Likelihood Estimator method available on the Fluctuation AnaLysis CalculatOR (FALCOR). The mutation rates of RIF r ranged from 9.24 × 10 −8 to 2.18 × 10 −10 , INH r 1.2 × 10 −7 ‑1.20 × 10 −9 , STR r 2.77 × 10 −8 ‑5.31 × 10 −8 and KAN r 1.7 × 10 −8 mutations per cell division. Data obtained in this study provide mutation rate estimates to key antitubercular drugs at a range of concentrations while also validating a number of the frequent approaches for estimating mutation rates.