Mycobacterium tuberculosis cultures were subjected to DNA fingerprinting with IS6110- and polymorphic GC-rich sequence (PGRS)-containing probes. The PGRS banding patterns remained highly stable during multiple cultures of specimens from one disease episode (0.5% changed) and during transmission in patients with close contact (1.9% changed). Characteristic PGRS-restriction fragment length polymorphism motifs for different strain groupings may indicate distant evolutionary events leading to the differentiation of M. tuberculosis strain lineages.