A repetitive DNA from a wild-type Mycobacterium bovis isolate was cloned and characterized. The repeated segment was present in M. bovis and M. tuberculosis but was absent from the six other mycobacteria tested. Sequence analysis demonstrated that this repetitive element belonged to the polymorphic GC-rich repeat sequence type, a family of interspersed repeated DNA. This fragment, when used as a probe in restriction fragment length polymorphism analyses, was able to detect polymorphism in M. bovis genotypes that went undetected when the established IS6110 was used as a probe. This repetitive element should be useful in epidemiological studies of bovine tuberculosis.