A total of 129 Mycobacterium bovis strains from 5 different Spanish locations were fingerprinted using the IS6110 repetitive element. We demonstrated the presence of multiple copies (from 2 to 13) of IS6110 in a large proportion (47.4%) of the M.bovis strains isolated from cattle and we showed that these strains can be successfully differentiated by means of the RFLP with IS6110. All of the M. bovis strains isolated from goats had multiple copies of IS6110 and 4 bands of 2, 1.7, 1.4 and 1.3 kb were common in all the caprine RFLP patterns. The caprine strains formed a clearly separate cluster from the bovine strains.