Mycobacterium bovis is the causative agent of bovine tuberculosis an important zoonose that is transmissible through the air or through the ingestion of milk and crude milk derivative products from sick animals. Conventional culture and biochemical tests remain the gold standard methodologies for detection of M. bovis in clinical samples as cattle tissues, nasal mucus, milk, blood and in environmental samples. Partly due to close genetic relatedness and variable biochemical patterns, definitive detection of M. bovis, up to species level, is time consuming and difficult. In this work, our objective was to design and validate a rapid and safe method for unequivocal detection of M. bovis in milk by PRA-PCR. The methodology was validated using genomic DNA extracted using the thermal lysis method from strains H37rv and H37ra of M. tuberculosis and strains of M. bovis. Primers tb11 (5'-ACCAACGATGGTGTGTCCAT-3’) and tb12 (5'-CTTGTCGAACCGCATACCCT-3’) were used to amplify a 441bp fragment from HSP65 gene. The amplified sequence was digested with Bste II (60ºC) and Hae III (37º) and the profile was used for footprinting species pertaining to the tuberculosis complex. A second confirmatory PCR performed with the primers jb21:(5'-TCGTCCGCTGATGCAAGTGC–3’) jb22: (5'-TCGTCCGCTGATGCAAGTGC-3') amplified a region of the RvD1-Rv203c gene (500pb) present only in M. bovis and M. bovis BCG genome. In a second step, raw milk was artificially contaminated with 10⁰ to 10⁸ cell/mL of M. bovis and the DNA was extracted with phenol:chloroform:alcohol isoamilic and precipitated with isopropanol after digestion of the whole milk with lysozyme, and proteinase K. The gene HSP65 amplicon (441bp) was obtained and after digestion presented the profile of characteristic footprinting for tuberculosis complex members 245pb, 125pb, 80pb with Bste II and 160pb, 140pb, 80pb with Hae III. By the same way, the second PCR using jb21 and jb22 primers amplified the fragment of 500bp. The detection limit of mycobacteria was 10 tubercle bacilli (in 1mL of milk). PRA-PCR methodology joins advantages such as quickness, high specificity and sensibility and can be applied in the inquiry of milk flocks affected by tuberculosis thus assisting the certification of free properties of this disease.