The molecular ecology of Mycobacterium avium subspecies paratuberculosis in the U.S. is poorly characterized. A discriminatory, reproducible method of DNA fingerprinting is needed to elucidate the clonal diversity of M. paratuberculosis. However, current methods of DNA fingerprinting M. paratuberculosis are generally labor-intensive, relatively slow, and do not yield high discriminatory power. The development of amplified fragment length polymorphism (AFLP) in a commercially available kit has introduced a fast, reproducible, highly discriminatory method for DNA fingerprinting. AFLP uses restriction digestion of the whole genome with two enzymes, ligation of adaptors to create unique ends, a non-selective PCR amplification, and a selective PCR amplification using fluorescent dye-labeled primers resulting in a completely computerized output. Objectives of this study were to standardize AFLP for use on M. paratuberculosis and compare it to a currently available method, multiplex PCR using IS900 (MPIL). Twenty-seven bovine isolates of M. paratuberculosis on agar slants from a small, Johne's-infected herd in Pennsylvania, U.S.A, were received and grown again in liquid culture. DNA was extracted using enzymatic lysis and standard phenol-chloroform extraction methods. MPIL was modified from the previously published protocol. Three selective primer combinations were ascertained to be optimal for AFLP, they are named A, B, and C here. Using MPIL, all 27 isolates belonged to the M1 subtype. For AFLP, dendrograms were created using the Dice coefficient, UPGMA, and the similarity cut-off set at 95%. A, B, and C individually yielded six, five, and eight different profiles, respectively, for the 27 isolates. The concordance values are good for comparisons between each primer combination. In conclusion, AFLP was faster, easier to use, and more discriminatory for DNA fingerprinting of M. paratuberculosis compared to MPIL for the isolates in this study.