Typing of Mycobacterium avium subspecies paratuberculosis strains presents a challenge since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole genome sequencing has extended possibilities for the characterization of Mycobacterium avium subspecies paratuberculosis and it can provide a phylogenetic context to facilitate global epidemiology studies. In this study we developed a SNP assay based on polymerase chain reaction and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133 Mycobacterium avium subspecies paratuberculosis isolates with different genotypes from eight different host species and seventeen distinct geographic regions around the world. A total of 28402 SNPs were identified among all the isolates. The minimum number of SNPs required to distinguish between all the 133 genomes was 93 and between only the Type C isolates was 41. To reduce the number of SNPs and PCRs required we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPs Mycobacterium avium subspecies paratuberculosis isolates can be characterized within 14 phylogenetic groups with a higher discriminatory power compared to MIRU-VNTR assay and other typing methods. Continuous updating of genome sequences are needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing of Mycobacterium avium subspecies paratuberculosis isolates.