Bovine tuberculosis is a prevalent zoonotic disease that causes high risks for production animals, dairy producers and consumers, together with significant economic losses. Thus, methods for easy, fast and specific detection ofMycobacterium bovisin small and medium-sized livestock under field conditions are very required. In this work, a Loop-Mediated Isothermal Amplification LAMP-PCR targeting the Region of Difference 12 (RD12) ofM. bovisgenome was designed for the purpose of identification. A set of six primers designed for the isothermal amplification of five different genomic fragments led to the specific identification ofM. bovisfrom other mycobacterial species. A basic colorimetric reaction was clearly observed at first sight under natural light, indicating positive identification ofM. bovisin a maximum of 30 min of isothermal amplification at 65 °C. The limit of detection was near 50 fg ofM. bovisgenomic DNA, corresponding approximately to 10 copies of the genome.•The proposed LAMP-PCR amplification of M. bovis genomic DNA might be performed by untrained laboratory personnel.•Specific identification of M. bovis LAMP is possible in 30 min at 65.. C using a simple water bath.•The basic colorimetric reaction for M. bovis identification could be observed with the naked eye under natural light.