The main causative agent of bovine tuberculosis (bTB), a livestock wordwide-spread but also zoonotic disease, is Mycobacterium bovis (M. bovis). The insertion sequence IS6110 is a genetic target frequently used for the molecular diagnosis of bTB. IS6110 may play a role in the phenotypic variation within the Mycobacterium tuberculosis complex (MTBC), an important pathogenic group to which M. bovis belongs. In contrast to other MTBC species, M. bovis is mostly considered as possessing one or very few copies of IS6110. Surprisingly, using a real-time IS6110-PCR as a first line diagnosis method for bTB in cattle, we observed variable detection signals depending on the genotype (spoligotyping-VNTR) of the infective strain, suggesting that IS6110 copy number could be variable among them.