Using molecular techniques, specifically, DNA-DNA hybridization in solution and measurement of the thermal stabilities of hybrids, we examined the genetic relationships among mycobactin-dependent mycobacteria and between such organisms and other (mycobactin-independent) mycobacteria. The mycobactin-dependent mycobacteria examined included five strains of Mycobacterium paratuberculosis, two wood pigeon isolates, and one bovine isolate that was biochemically identified as Mycobacterium avium. All mycobactin-dependent mycobacteria were found to belong to the same hybridization group and to be closely related to organisms in M. avium complex serovars 2, 8, and 9, but more distantly related to organisms in serovars 7 and 19. Relatively low levels of hybridization were observed with other mycobacterial species examined. Thus, these results provide genetic evidence to support previous biochemical and cultural evidence that indicated that mycobactin-dependent mycobacteria constitute a closely related group of organisms within the M. avium complex.