Defining genetic diversity in the wake of the release of several Mycobacterium avium subsp. paratuberculosis (MAP) genome sequences has become a major emphasis in the molecular biology and epidemiology of Johne's disease research. These data can now be used to define the extent of strain diversity on the farm. However, to perform these important tasks, researchers must have a way to distinguish the many MAP isolates/strains that are present in the environment or host to enable tracking over time. Recent studies have described genetic diversity of the Mycobacterium avium complex (MAC), of which MAP is a member, through pulsed-field gel electrophoresis, single sequence repeats, variable-number tandem repeats, genome rearrangements, single nucleotide polymorphisms and genomewide comparisons to identify insertions and deletions. Combinations of these methods can now provide discrimination sufficient for dependable strain tracking. These molecular epidemiology techniques are being applied to understand transmission of Johne's disease within dairy cattle herds as well as identify which strains predominate in wildlife.