A recently developed lateral flow immunochromatographic device (LFD) for detection of Mycobacterium bovis cells was comprehensively evaluated for applications in veterinary diagnostic laboratories in different parts of the world. The LFD specifically detected M. bovis in the broadest sense (var bovis, var caprae and BCG) and, of a wide range of Mycobacterium spp. tested, cross-reacted only with one strain of M. pinnepedii. Analytical sensitivity and specificity of the novel LFD were excellent when the test was used to confirm the presence of M. bovis in MGIT liquid cultures or the identity of suspect colonies isolated on Coletsos, Lowenstein-Jensen or Stonebrink media from a variety of veterinary specimens. Although some bovine lymph node homogenates tested positive for M. bovis by the LFD, the test lacks sufficient analytical sensitivity to be a rapid method to directly detect M. bovis in veterinary diagnostic samples without prior culture. There was significant correlation between results obtained with the commercially available BD MGIT™ TBc ID test and the novel M. bovis-specific LFD (r = 0.9791; P<0.0001) applied to 190 MGIT cultures of bovine specimens, with the added benefit that the novel LFD differentiated M. bovis from another member of the M. tuberculosis complex in one positive culture. This multi-laboratory evaluation of the novel LFD test has clearly demonstrated the assay’s unique specificity for M. bovis and its potential utility as a rapid and inexpensive test to confirm isolation of M. bovis from veterinary specimens following liquid culture, or to confirm suspect colonies after solid culture.