Abstract: Tuberculosis (TB) is a zoonotic disease affecting mammals worldwide. Herbivore TB is caused by Mycobacterium bovis and other Nontuberculous Mycobacteria (NTM). This study aimed to investigate the prevalence of mycobacterial species and to evaluate the diagnostic methods in detecting mycobacterial infection in Intradermal Tuberculin Test (ITT) positive animal. Samples were collected from ITT positive animals (151 cattle and 36 deer) for mycobacterial isolation, pathological examination, acid-fast stain, molecular diagnosis and ELISA. The detection rate of mycobacterial infection in tissues differed among methods from highest in gross lesion (105, 69.5%), Mn-PCR (89, 64.2%), histopathology (89, 58.9%) to acid fast acid (84, 55.6%) in cattle and from Mn-PCR (28, 77.8%), gross lesion and histopathology (19, 52.8%), to acid fast stain (47.2%) in deer. Among 64 culture positive, 48 samples were infected by M. bovis (30 in cow vs. 18 in deer). ELISA examination found high level of M. paratuberculosis infection (67, 44.4%) in cattle and M. bovis (18, 52.8%) in deer. Among gross lesion, histopathology, acid fast stain and Mn-PCR methods, the most prevalent tissues of mycobacterial infection were retropharynx lymph node and mediastinal lymph nodes in cattle and retropharynx lymph node and lung in deer. Farm with only once ITT in routine test may be not true M. bovis infected and ITT was overestimated in national MTB clearance program and the reliability in Taiwan. DNA sequence analysis of M. bovis demonstrated identical genotypes between deer and cattle from Yulin and diverse origins among cattle from three counties.