Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis (Johne′s disease) in ruminants. The infected and carrier animals shed the microorganism intermittently in faeces. In order to ensure the sensitive identification of MAP-shedders by the examination of faecal samples. Real-time PCR assays were compared which amplify the insertion sequences IS900 and ISMAV2 and furthermore the genomic element F57. The assays were designed as duplex-PCR including the amplification of PUC19-plasmid as internal control. The analytical sensitivity of the assays was determined using DNA of 6 different isolates of MAP in broad linear range (50 ng-5 fg μL–1). The specificity was validated using 23 known species and subspecies of the Mycobacteriacea and 18 other non-Mycobacteriacea pathogens. The sensitivity for detection of MAP-DNA was 5 fg/ reaction targeting IS900. Reproducible detection limit for real-time PCR targeting ISMAV2 and F57 was 50 fg reaction. All Mycobacteriacea different from MAP and non-Mycobacteriacea gave negative results for ISMAV2 and F57 sequence. For IS900 weak positive signals were observed with highly concentrated DNA (5 ng μL–1) of 3 Mycobacterium avium subsp. avium strains from cattle and poultry but not with low concentrated DNA (5 fg μL–1). Thus false-positive results should not be found if analyzing ruminant faeces directly with IS900-PCR. ISMAV2-PCR and F57-PCR has to be preferred to IS900-PCR if real-time PCR is intended for the specification of cultured Mycobacteria.