We present a case of tuberculosis of the pancreas in a 65 year-old man diagnosed by detection of Mycobacterium tuberculosis DNA in a resection specimen using a highly specific polymerase chain reaction-based assay. Almost all pre-operative and intra-operative findings except for helical computed tomography were highly suggestive of malignant pancreatic tumor. However, the histopathology of the pancreas revealed caseating granulomatous inflammation of possible tuberculous etiology, although special staining and culture of these tissues gave negative results. Only detection of Mycobacterium tuberculosis DNA using the polymerase chain reaction-based assay gave a positive result which allowed antituberculosis treatment to be started. The patient's post-operative recovery has been uneventful without complications. If an early accurate diagnosis can be made in such cases, antituberculosis treatment can be started rapidly.