Background and aims: Conflicting results exist about the presence of Mycobacterium avium subspecies paratuberculosis (MAP) specific IS900 DNA in Crohn’s disease (CD) tissues. Therefore, we examined IS900 in a large number of gut samples from patients with CD (n = 100) and ulcerative colitis (UC, n = 100), and in non-inflamed control tissues (nIBD, n = 100). We hypothesised that IS900 DNA detection might be associated with distinct clinical phenotypic characteristics in CD. Methods: The prevalence of MAP DNA in surgically resected tissues was examined using a mechanical-enzymatic disruption technique and nested IS900 specific polymerase chain reaction (PCR). CD patients were stratified according to the criteria of the Vienna classification and other clinical characteristics. Results: IS900 PCR detection rate was significantly higher in CD tissue samples (52%) than in UC (2%) or nIBD (5%) specimens (p < 0.0001 ). In CD patients, IS900 DNA was detected in samples from both diseased small bowel (47%) as well as from the colon (61%). No firm association between MAP specific IS900 detection rates and clinical phenotypic characteristics in CD could be established. However, corticosteroid medication constituted a factor which tended to have a negative influence on IS900 DNA detection rates in CD(p < 0.01). Conclusions: The presence of MAP specific IS900 DNA is a predominant feature of CD. Therapeutic intervention against MAP might represent a potential target for disease mitigation in Crohn’s disease. Background and aims: Conflicting results exist about the presence of Mycobacterium avium subspecies paratuberculosis (MAP) specific IS900 DNA in Crohn’s disease (CD) tissues. Thus, we examined IS900 in a large number of gut samples from patients with CD (n = 100) and ulcerative colitis (UC n = 100), and in non-inflamed control tissues (nIBD, n = 100). We hypothesised that IS900 DNA detection might be associated with distinct clinical phenotypic characteristics in CD. Methods: The prevalence of MAP DNA in surgically resected tissues was examined using a mechanical-enzymatic disruption technique and nested IS900 specific polymerase chain reaction (PCR). CD patients were stratified according to the criteria of the Vienna classification and other clinical characteristics. Results: IS900 PCR detection rate was significantly higher in CD patients, IS900 DNA was detected in samples from both diseased small (52%) than in UC (2%) or nIBD (5%) specimens However, corticosteroid could have established a negative influence on bowel (47%) as well as from the colon (61%). No firm association between MAP specific IS900 detection rates and clinical phenotypic characteristics in CD could be established. However, Conclusions: The presence of MAP specific IS900 DNA is a predominant feature of CD. Therapeutic intervention against MAP might represent a potential target for disease mitigation in Crohn’s disease.