Recent studies have shown a difference in the genotype of resistant bacteria following passage in animals compared to those passaged in vitro. This suggests that organisms rapidly adapt to their environment of growth. We sought to investigate whether this phenomenon occurred in human infection and whether changes could be detected in the fitness (growth velocity) of isolates transmitted between human hosts. Isogenic strains of Mycobacterium tuberculosis were obtained from a well-documented hospital outbreak. The subjects included those who were HIV seropositive and immunocompromised. The relative fitness of each sample was measured using growth competition in vitro. The results confirmed that our method of measuring fitness was not influenced by the storage conditions of the isolates, and demonstrated that the fitness of genetically similar isolates obtained from different patients in the outbreak differed significantly, as reflected in the growth velocity of the strains. This study provides the first evidence that multiple drug resistant M. tuberculosis strains adapt to the environment of their human host.