Acquisition of antibiotic resistance in Mycobacterium tuberculosis has been shown to incur a fitness cost; the size of which depends on the position and nature of the mutation. The frequency at which resistant M. tuberculosis strains are isolated in vivo has been correlated with the in vitro fitness of the different corresponding mutants in laboratory strains. We have previously provided evidence that strains adapt in vivo to their host. To investigate the fitness cost of drug resistance in clinical strains we developed a rapid method of fitness estimation using liquid culture using a modification of the Youmans and Youmans method. Using this method we determined the generation time (G) of a wide range of clinical isolates. The G of rifampicin resistant clinical strains (mean 39.3 h) was longer than drug sensitive clinical strains (mean 19.50 h) which was in turn longer than laboratory strains (mean 15.19 h) (with a P value of 0.0065 using one way ANOVA with Kruskal-Wallis test for significance). Drug resistant strains, not including those resistant to rifampicin did not appear to have an increased generation time (mean 18.18 h) compared to drug sensitive clinical strains. The composite fitness deficient was influenced by the genetic mutation and the ability of the strain to adapt to the landscape of the human host.