Although the heterogeneity of Mycobacterium tuberculosis populations and the existence of mixed infections are now generally accepted, systematic studies on their relative importance are rare. In the present study, 10 individual colonies of each M. tuberculosis isolate (primary isolate) from 97 tuberculosis patients in a primarily human immunodeficiency virus-negative population were screened for heterogeneity and detectable mixed infections by spoligotyping, IS6110-based restriction fragment length polymorphism analysis, and mycobacterial interspersed repetitive unit-variable number of tandem repeat typing. The MICs of antituberculosis drugs for colonies with divergent fingerprints were determined. Infections with different bacterial subpopulations were detected in the samples from eight patients (8.2%), and the frequency of detectable mixed infections in the study population was estimated to be 2.1%. Genotypic variations were found to be independent of the drug susceptibilities, and the various molecular markers evolved independently in most cases. The predominant strains and the primary isolates always had concordant drug susceptibility and MIC testing results. These findings have implications on the interpretation of molecular epidemiology results for patient follow-up and in transmission studies.Tuberculosis (TB) patients have often been assumed to be infected with a single Mycobacterium tuberculosis strain, and infection with one strain is thought to confer immunity to additional M. tuberculosis infections. Therefore, a recurrence of disease after treatment is often considered to be caused by the same strain that caused the original infection. However, cases of reinfection by a second M. tuberculosis strain and occasional infection with more than one strain have been documented (2,5,12,22,31), and the occurrence of mixed infections has now become generally accepted. Infection with multiple strains of M. tuberculosis can seriously confuse interpretation of drug susceptibility testing (DST) results and the detection of epidemiological links (2, 44). However, the frequency of mixed infections and the appreciation of their impact on the phenotypic and the genotypic properties of the primary isolates largely remain unknown, essentially because of the lack of systematic studies on the heterogeneity within M. tuberculosis populations from an individual patient.Almost 30 years ago, Mankiewicz and Liivak (19) used phage typing to analyze the heterogeneity among individual colonies obtained from cultures of specimens isolated from 233 Eskimo patients, leading to the conclusion that 14.1% of the patients tested were simultaneously infected with more than one M. tuberculosis strain. The development of DNA fingerprinting tools has, meanwhile, considerably improved the capacity to distinguish M. tuberculosis strains. Among these tools, IS6110-based restriction fragment length polymorphism (IS6110-RFLP) analysis is considered the standard technique for comparison of M. tuberculosis isolates (32, 33). Other techniques, su...