The analysis of the gyrA and gyrB genes of a panel of Mycobacterium avium subsp. paratuberculosis isolates from types I, II, and III detected type-specific single nucleotide polymorphisms. Based on these results, we developed a PCR and restriction enzyme analysis to discriminate type I and III isolates. The application of this technique would be the unique strategy to characterize these strains when there is not enough bacterial growth to perform pulsed-field gel electrophoresis and IS900 restriction fragment length polymorphism.