The insertion sequence IS900 has long been utilized as a genetic marker specific for Mycobacterium avium subsp. paratuberculosis (Map). However, recent studies have found that IS900 or closely related sequences may also be present in M. avium subsp. avium (Maa). Fibronectin attachment protein (FAP) has been shown to be required for the attachment of Map to fibronectio. The fap genes in Map and Maa are genetically similar. The objective of the present study was to exploit nucleotide sequences of fap in M. avium subspecies for detection and differentiation of Map from other subspecies of M. avium. A pair of oligonucleotide primers was designed to flank a region near the C-terminus of fap from Map and Maa. These primers were used to amplify DNA extracted from laboratory and field strains of Map, Maa, and M. avium subsp. Amplicons were sequenced from selected M. avium isolates. Electrophoretic migration patterns were compared following restriction digestion of Map amplicons. All Map strains yielded a 151-bp product that produced identical migration patterns when digested with either AscI or ApaI. Sequenced Map amplicons shared the same identity as the GenBank reference sequence. In contrast, Maa isolates produced 178, 166, or 118-bp PCR products, the latter two of which were apparent truncations of the 178-bp amplicon. The results revealed that PCR targeting fap can reliably distinguish Map from other M. avium subspecies, thus indicating its usefulness as a diagnostic tool.