Restriction enzyme fingerprinting of genomic DNA and Southern blots probed with subclones of the Mycoplasma pneumoniae cytadhesin P1 gene were used to characterize clinical isolates of M. pneumoniae. On the basis of the examination of 29 individual M. pneumoniae isolates, two distinct groups were established. Group 1, which displayed a 12-kilobase band following DNA digestion with HindIII, consisted of strain M129-B16 and three others obtained in the state of Washington during the 1960s. The remaining M. pneumoniae strains belonged to group 2, which lacked the 12-kilobase band and included samples from the 1940s, 1970s, and 1980s. This category also included the only M. pneumoniae strain isolated from the synovial fluid of an arthritic patient.