Tuberculosis and Crohn disease are granulomatous disorders affecting the intestinal tract with similar clinical manifestations and pathologic features. We evaluated the use of in situ polymerase chain reaction (PCR) using Mycobacterium tuberculosis complex–specific primers for IS6110 to differentiate these 2 disorders in archival mucosal biopsy specimens. In situ PCR was positive in 6 of 20 tuberculosis biopsy specimens and 1 of 20 Crohn disease biopsy specimens. Staining was localized to a site of granulomatous inflammation in 3 of the tuberculosis specimens and in the Crohn disease specimen. In the other tuberculosis biopsy specimens, positive staining was localized to inflammatory granulation tissue and to a focus of intact mucosa without granulomatous inflammation. The presence of M tuberculosis DNA in Crohn disease could be due to coexisting latent tuberculosis or indicate a role for these bacteria in triggering an abnormal immune response. Therefore, in situ PCR is potentially useful to differentiate intestinal tuberculosis from Crohn disease, if the sensitivity is improved.