Summary Drug resistance in Mycobacteriumtuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug-resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in M. smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A-site of 16S rRNA that (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem-loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug-resistant tuberculosis disease.