Bovine tuberculosis (TB) caused byMycobacterium bovisis a significant health threat to cattle and a zoonotic threat for humans in many developing countries. Rapid and accurate detection ofM.bovisis fundamental for controlling the disease in animals and humans, and for the proper treatment of patients as one of the first-line anti-TB drug, pyrazinamide, is ineffective againstM.bovis. Currently, there are no rapid, simplified and low-cost diagnostic methods that can be easily integrated for use in many developing countries. Here, we report the development of a loop-mediated isothermal amplification (LAMP) assay for specific identification ofM.bovisby targeting the region of difference 4 (RD4), a 12.7 kb genomic region that is deleted solely inM.bovis. The assay's specificity was evaluated using 139 isolates comprising 65M.bovisisolates, 40M.tuberculosisisolates, sevenM.tuberculosiscomplex reference strains, 22 non-tuberculous mycobacteria and five other bacteria. The established LAMP detected onlyM.bovisisolates as positive and no false positives were observed using the other mycobacteria and non-mycobacteria tested. Our LAMP assay detected as low as 10 copies ofM.bovisgenomic DNA within 40 minutes. The procedure of LAMP is simple with an incubation at a constant temperature. Results are observed with the naked eye by a color change, and there is no need for expensive equipment. The established LAMP can be used for the detection ofM.bovisinfections in cattle and humans in resource-limited areas.