Aims: To develop an antibody broadly reactive against mycobacterial species, which will improve detection of mycobacteria in tissue sections by immunohistochemistry (IHC). Methods: A sheep antisera was developed by immunization with multiple mycobacteria, and was tested by IHC against a range of mycobacteria in tissues from many species, as well as negative tissue controls and other bacteria. Results: The sheep antiserum, MAS-01, reacted with all 18 mycobacterial species tested, but did not react with uninfected inflammatory tissues. Although MAS-01 cross-reacted with two microbial genera which are related to mycobacteria (Corynebacteria and Proprionibacteria), it did not with Nocardia or Actinomyces. The antibody was more sensitive than the Ziehl-Neelsen stain for detection of tissue mycobacteria, and shortened the time required to identify these infections. Conclusion: The MAS-01 antiserum will facilitate rapid identification of tissue mycobacterial infection by histopathologists.