Two hundred three freeze-dried strains of Mycobacterium tuberculosis collected during the 1960s were compared with 4102 strains collected during the 1990s, and 14 DNA patterns identified among the “historical strains” were 100% identical to patterns identified among the “recent strains.” They were isolated from 41 and 40 patients who had tuberculosis during the 1960s and 1990s, respectively. The patients’ mean age differed by >30 years, a finding strongly suggesting that the patients from the 1990s experienced reactivation of M. tuberculosis infection acquired during the 1960s. The half-life of IS6110 DNA patterns during latency was estimated to be 36 years (95% confidence interval, 25–54 years). Thus, this comparison of historical and recent strains yields molecular epidemiologic evidence of M. tuberculosis reactivation spanning decades and suggests that the rate of change of DNA patterns during latency is much longer than that during active disease. This has important implications for the interpretation of clustering, especially for the extent of recent transmission