The reservoir and transmission route of Mycobacterium kansasii are largely unknown. In addition, culturing of M. kansasii from human sources is not proof of disease because it may represent colonization rather than infection. Unfortunately, investigation of the epidemiology and pathogenicity of M. kansasii is complicated by evidence of heterogeneity within the species. A comprehensive study by detailed genotypic analysis of a large collection of M. kansasii isolates (n = 276) from various geographical sources within Europe was conducted. Five defined subtypes of M. kansasii were identified; of these subtypes, type I represents the most common isolate from humans. Although phylogenetic analysis confirmed its relationship to the other M. kansasii types, significant sequence divergence was found at the 16S-23S intergenic spacer. Analysis of the chromosomal polymorphism of type I demonstrated a marked clonal structure for this particular organism. Because M. kansasii is becoming a significant pathogen among immunodeficient hosts, future epidemiological and pathogenicity studies should take into consideration both the heterogeneity within the species and the apparent clonality of the most prevalent M. kansasii isolates infecting humans.