Tuberculosis is still one of the most important infectious diseases worldwide. Although human cases due to Mycobacterium bovis, the aetiological agent of bovine tuberculosis (bTB), considerably decreased in most industrialized countries, the possible zoonotic health hazard and the considerable economic losses it brings justify significant efforts to eradicate the disease in several countries. In Portugal, although a systematic slaughter policy of tuberculin reactive animals has been ongoing for several years, eradication is far from being achieved. In order to clarify possible infection sources and transmission routes, molecular typing of Portuguese M. bovis animal strains was undertaken. Two typing methods directed on potentially polymorphic genomic regions were chosen: Spoligotyping, a reverse line blot hybridization technique that evaluates the presence or absence of 43 oligonucleotide sequences in the Direct Repeat genomic region, and MIRU-VNTR (Mycobacterial Interspersed Repetitive Units- Variable Number Tandem Repeats) typing of 8 selected minisatellite like loci.