OBJECTIVE Mycobacterium avium subspecies paratuberculosis (MAP) has been reported in Iranian cattle since 1965. Little is known on the population genetics of MAP in this Middle-Eastern State. A principle scope of this study was therefore genetic characterization of MAP isolates collected from farm animals in Iran. METHODS Sixteen field isolates of MAP collected from bovine (n=8), ovine (n=3), and caprine (n=2) hosts in Alborz, Fars, Isfahan, Qazvin, Tehran, and Zanjan provinces were subcultured on mycobactin J-supplemented Herrold's egg slopes in order to provide the required genomic material. The laboratory strain MAP III & V was included as the reference strain. IS900-RFLP (Restriction Fragment length Polymorphism) was conducted using BsteII restriction enzyme. Application of IS900-RFLP genotyping on 16 Iranian MAP isolates in the present study classified them into six observable but similar types represented by two clustered and four orphan types. The laboratory strain MAP III & V displayed a totally different pattern easily distinguishable from that of Iranians. RESULTS Detection of six genotypes among 16 wild isolates is an indication of a population with a potentially high level of diversity. We assume that, with inclusion of more field isolates, it is very likely that even higher diversity may be observed within the studied isolates. The different patterns displayed by the Iranians and the laboratory strain in this work might explain the independent evolutionary pathways these have gone through to evolve from their ancestral clones. CONCLUSIONS Further description on population genetic of MAP in Iran urges for more epidemiological work using similar and alternative standard genotyping system.