Mycobacterium tuberculosis isolates from multiple drug resistant or extensively drug resistant patients show a particular set of mutations in drug targets conferring resistance. However, the selection of drug-resistant strains in vitro yields an alternative set of mutations, thought to result from the cost-benefit associated with drug resistance. Mutations allowing for survival under antibiotic may not be beneficial when presented with the host environment or with a drug-free environment. These fitness effects drive the natural evolution of this bacterium. Using recombineering a large cohort of mutations was generated within two drug targets, inhA and gyrA, to study in vitro the variability of mutations allowable under either isoniazid or ofloxacin, respectively. As a proof of concept this process was carried out in Mycobacterium smegmatis. Analysis of survivors allowed for identification of novel mutations and substitutions, as well as showing mutations previously found only in clinical isolates can be present in laboratory isolates.