Clinical trials are underway to evaluate the potential of fluoroquinolones to shorten the standard tuberculosis treatment, and new fluoroquinolone derivatives are being developed aiming at higher specificity and activity against Mycobacterium tuberculosis. The emergence of drug resistance represents the most significant threat to the success of this new class of anti-tuberculosis agents. Theoretical and experimental data suggest that this process will significantly be influenced by differences in bacterial fitness. We recently showed that the competitive fitness of rifampin-resistant M. tuberculosis depends on the specific resistance-conferring mutation and strain genetic background, and that competitive fitness experiments are predicative of the epidemiology of drug resistance in clinical settings. Here we propose to explore these phenomena in fluoroquinolones. We will generate spontaneous fluoroquinolone-resistant strains in two different strain backgrounds and measure their competitive fitness. We will compare these fitness measurements to the frequency of the corresponding resistance alleles in a collection of clinical fluoroquinolone-resistant strains, which we will compile through our international collaborations. We will experimentally evolve some of the fluoroquinolone- resistant mutants to allow for compensation, and use comparative whole-genome resequencing to identify putative compensatory mutations. These putative compensatory mutations will be validated in our collection of clinical fluoroquinolone-resistant strains and, as a control, in our collection of pan-susceptible strains representative of the global phylogenetic diversity of M. tuberculosis. This project represents a first step towards a better understanding of the ecology of fluoroquinolone resistance in M. tuberculosis, with potentially important implications for tuberculosis control and drug development. Subsequent work will then be able to identify the molecular basis of fitness differences and compensation. New drugs are urgently needed for the treatment of tuberculosis. A new class of antimicrobials, the fluoroquinolones, are promising candidates that are currently being investigated. However, resistance to these new drugs is likely to emerge. In order to maximize the benefit for tuberculosis control, there is an urgent need to better understand the bacterial factors that influence resistance to fluoroquinolones.