Introduction and objectives: 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. Thanks to the development of sequencing technologies, the number of available Map genomes is increasing rapidly, which makes it possible to analyze the distribution of IS900 in this very slow-growing bacterium. The objective of this study was to use bioinformatics approaches to study IS900 distribution in the genomes of Map strains and to develop automated in silico IS900 restriction fragment length polymorphism (IS900-RFLP) analysis. Materials and methods: The complete genomes of the strains K-10, Telford and JII-1961 were chosen to cover the genetic diversity of Map divided into two strain types, S and C, according to the phylogeny of this species. Computer analysis included software located IS900 using BLAST and determined fragments from complete genome (FASTA) using BioPython. Digital representation was provided using matplotlib. Profile comparisons were carried out using Bionumerics software. Results, discussion and conclusion: The program developed in this study allowed automated location of IS900 sequences to identify their positions and their number. Between 17 to 22 copies of the IS900 sequence were found in the genomes studied. An in silico RFLP analysis was developed using complete genomes. This procedure utilised the BstEII restriction site conventionally used for Map to obtain the exact size of the DNA fragments carrying a copy of the IS900 sequence. IS900 RFLP profiles were compared using fragment sizes or by digital visualization of the restriction fragments separated according to their size by mimicking their migration in agarose gel. These digital profiles were compared to the many existing profiles in the literature. 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.