Histopathology is used as the definitive diagnostic test for ovine Johne's disease in Australian sheep flocks. The cost of this diagnostic method is a major limitation on its more widespread use. These costs can be reduced significantly by decreasing the number of tissues submitted for processing to those vital for diagnosis by this means. The Australian Standard Diagnostic Technique (ASDT) nominates that ileum (3 sites, 1 metre apart, the first being adjacent to the ileocaecal valve), colon, caudal jejunal lymph node and ileocaecal lymph node (1 site each) must be sampled from each sheep submitted for diagnosis of Johne's disease Tissues from 400 sheep with histological evidence of ovine Johne's disease were studied. These originated from sheep either selected by the owner as displaying clinical Johne's disease and submitted to the Regional Veterinary Laboratory, Orange for necropsy (n=250), or from formalin fixed tissues submitted following field necropsy of animals selected on the basis of positive serology (Agar Gel Immunodiffusion Test) in a surveillance testing program (n=150). The frequency of positive histological lesions and the presence of acid-fast bacilli (AFB) for the tissues nominated by the ASDT within this population are reported. All sheep had diagnostic histological lesions in either the terminal ileum (adjacent to the ileo-caecal valve) or the caudal jejunal lymph node. Examination of additional tissues was not warranted and only increased the cost of histological diagnosis. While widespread sampling may be necessary to detect the very early stages of infection by histological means, the additional sites nominated in the ASDT are unlikely to enhance the sensitivity of histological diagnosis.