DNA fingerprints were generated using PFGE following extraction of mycobacterial DNA and digestion with the restriction endonucleases Xba-1 and Vsp-1 for many of the known human paratuberculosis isolates (Holland, Holland-1, Holland-2, Linda, Dominic and Ben) and the type strain ATCC 19698. Preliminary results indicate that three isolates (Linda, Dominic, Holland-1 and the type strain) demonstrated genetically identical PFGE fingerprint patterns when cut with either Xba-1 or Vsp-1. Fewer bands were observed with Vsp-1 compared with Xba-1, but both were useful in determining whether strains were similar or different. All strains could be placed into 4 distinct groups based on their PFGE fingerprint patterns. In addition, we have used PFGE to examine a number of M. paratuberculosis isolates from different animals. An isolate from a Wisconsin farm demonstrated the same identical PFGE pattern as an isolate obtained from a Florida Zoo using the enzyme Xba-1, whereas, there was a single band difference between these same isolates with Vsp-1. We conclude that PFGE is a useful tool in differentiating genotypic relationships among M. paratuberculosis and is helpful in providing evidence for strains being epidemiological linked.