We studied the population genetics of Mycobacterium kansasii isolates from the United States by PCR restriction enzyme analysis (PRA) of the 441-bp Telenti fragment of the hsp-65 gene and pulsed-field gel electrophoresis (PFGE) of genomic DNA with the restriction endonucleases AseI, DraI, and XbaI, and we compared the patterns to those previously reported from France and Japan. By PRA, 78 of 81 clinical isolates (96%) from the United States belonged to subspecies I. With PFGE, 28 AseI patterns, 32 DraI patterns, and 35 XbaI patterns were produced. PFGE showed marked clonality of the U.S. isolates, with differences between genotypes involving only one or two bands. Isolates within Texas showed lower pattern diversity than those from different states. With DraI, 31 of 71 isolates (44%) had the same common PFGE pattern, which matched the predominant pattern in France (pattern Ia), determined by Picardeau et al. (M. Picardeau, G. Prod'hom, L. Raskine, M. P. LePennec, and V. Vincent, J. Clin. Microbiol. 35:25-32, 1997), and in Japan (type M), determined by Iinuma et al. (Y. Iinuma, S. Ichiyama, Y. Hasegawa, K. Shimokata, S. Kawahara, and T. Matsushima, J. Clin. Microbiol. 35:596-599, 1997). With AseI, 42% of isolates produced a common pattern indistinguishable from the common pattern seen in French isolates (Ia) and with only one band difference from the common pattern (type M) in Japan. This study demonstrates that subspecies I is the predominant subspecies of M. kansasii among clinical isolates in the United States, as it is in Europe and Japan, and that genotype I is highly clonal worldwide, with the same major genotype responsible for human infection. The fact that a single clone of M. kansasii is responsible for most cases of human disease suggests that specific virulence factors may be associated with this specific genotype.