Mycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis or Johne’s disease that causes chronic intestinal inflammation in ruminants. The IS900 insertion sequence, specific for Map, has been used widely as an epidemiological marker and target for qPCR diagnosis. Recently, the complete genomes of strains belonging to the major phylogenetic lineages of Map have been made available. This has allowed us to investigate the distribution of IS900 in this slow growing bacterium. The objective of this study is to characterize the distribution of the IS900 element and how it affects genomic evolution and gene function of Map. A secondary goal is to develop automated in silico restriction fragment length polymorphism (RFLP) analysis using IS900. Complete genomes from C-type and S-type (including subtypes I and III) lineages were chosen to represent the genetic diversity of Map. Computer analysis included software located IS900 using BLAST and determined fragments from complete genome (FASTA) developed with Biopython. Digital representation was provided using matplotlib. Profile comparisons were carried out using Bionumerics software. Upstream and downstream genomic regions flanking each IS900 copy were extracted and used to identify orthologous insertion site across genomes. The program developed in this study allowed automated location of IS900 sequences. Between 16 to 22 copies of the IS900 sequence were found in the genomes studied. Nine IS900 insertion site locations were conserved across all genomes studied while smaller subsets were unique to a particular lineage. An in silico IS900 RFLP analysis was developed and profiles were compared by digital visualization. This study provided a program making it possible to automate IS900 distribution analysis in Map genomes to enrich our knowledge on the dynamics of distribution of this IS for epidemiological purposes, for understanding the evolution within the Map species and studying the biological implication of the presence of IS900.