AbstractMycobacteria population can undergo mutations in their DNA sequence during replication, which if not repaired, would be transferred to future generations. In this study, in vitro spontaneous mutations in Mycobacterium smegmatis mc2155 (Msm) conferring resistance to isoniazid (INHr), rifampicin (RIFr), kanamycin (KANr) and streptomycin (STRr) were determined at several concentrations in a fluctuation assay. Mutation rate was estimated using the P₀ method, and estimates were then compared with the Lea-Coulson method of the median and Ma-Sandri-Sarkar Maximum Likelihood Estimator (MSS-MLE) method available on the Fluctuation analysis calculator (FALCOR). The mutation rates of RIFr ranged from 9.24 × 10-8 - 2.18 × 10-10, INHr 1.2 × 10-7 - 1.20×10-9, STRr 2.77 × 10-8 - 5.31 × 10-8 and KANr 1.7 × 10-8 mutations per cell division. This study provides mutation rate estimates to key antitubercular drugs at a range of concentrations.