In an ongoing molecular epidemiology study, human immunodeficiency virus-negative patients with first-time pulmonary tuberculosis from a high-incidence community were enrolled. Mycobacterium tuberculosis strains were identified by restriction fragment length polymorphism analysis with two fingerprinting probes. Of 131 patients, 3 (2.3%) were shown to have a mixture of strains in one or two of their serial cultures. This study further investigated these cases with disease caused by multiple M. tuberculosis strains in the context of the molecular epidemiology of the study setting.