For molecular biological detection of Mycobacterium avium subsp. paratuberculosis (MAP), PCR methods with primers targeting different regions specific for MAP are used worldwide. However, some uncertainties exist concerning the specificity of certain target regions and the sensitivity. To identify the methods which are best suited for diagnostics, 8 single-round and 5 nested PCR systems including 12 different primer pairs based on IS900 (9 x), ISMav2 (1x), f57 (1x), and locus 255 (1x) sequences were compared regarding their analytical sensitivity and specificity under similar PCR conditions. Reference strains and field isolates of 17 Mycobacterium species and subspecies, 16 different non-mycobacterial bovine pathogens and commensals were included in this study. Single-round PCR resulted in a detection limit of 100 fg to 1 pg, and nested PCR in 10 fg or below. Depending on the specific primer sequences targeting IS900, false positive results occurred with one of the five single-round and two of the four nested PCR systems. This also applied to the single-round PCR based on ISMav2 and the nested PCR based on f57. A high number of non-specific products were primarily detected for the single-round PCR assay based on ISMav2, but also for a single-round PCR targeting the IS900 and the locus 255. In conclusion, stringent selection of IS900-specific primers ensures that IS900 remains a favourite target sequence for amplification of MAP specific loci. The studied PCR systems based on f57, and locus 255 can also be recommended. Revision of ISMav2 primers is necessary. Single-round PCR systems are very reliable. Nested PCR assays were occasionally disturbed by contaminations, thus bearing a risk for routine diagnostics.