To define the genetic diversity among Mycobacterium avium isolates from human immunodeficiency virus-infected patients, specimens were cultured prospectively, and isolates obtained from 14 patients (4 with positive blood, stool, and sputum; 6 with positive blood and stool; 3 with positive blood only; and 1 with positive stool only) were studied. Both serotyping and ribotyping had limited ability to discriminate among isolates from different patients, whereas the distinctive restriction fragment profiles resolved by pulsed-field gel electrophoresis indicated that each patient was infected by a unique strain. Of the 13 bacteremic patients, 2 were bacteremic concurrently with 2 distinct strains. The fact that M. avium isolates from AIDS patients exhibit considerable genetic diversity supports the hypothesis that the infection is acquired from various environmental sources. Further, individual patients are not infrequently bacteremic with > 1 strain simultaneously, which may need to be considered in protocols for the diagnosis and management of M. avium disease.