Paratuberculosis (Johne's disease) is a chronic granulomatous enteropathy of ruminants. It affects primarily cattle, sheep, and goats and is caused by Mycobacterium avium subsp. paratuberculosis (Map). Paratuberculosis is a common disease in many countries worldwide, and its effects on production can be economically significant. In Chile paratuberculosis has been reported in cattle, sheep and more recently it has been described in goats. Preliminary data suggests that the infection is also present in Chilean wild animals such as guanacos, as well as in some other introduced wild animal species. Nevertheless, there is a lack of molecular epidemiological data on the type of strains existing in Chile. A deep knowledge of paratuberculosis infection transmission between and within both domestic and wildlife host species should be the basis to set up a national control programme. The classification of Map isolates using genomic typing methods provides some understanding of the infection. A method that detects a stable variation at base pair 223 in the IS1311 using polymerase chain reaction with restriction endonuclease analysis (PCR-REA) provides a fast and easy way to differentiate between cattle and sheep Map strains. This IS1311 PCR-REA analysis was used to detect genetic differences among 28 Map isolates from cattle (19), goats (9), guanacos (3) and deer (1) from different regions of Chile. All isolates were C-type and probably of bovine origin. These results showed no genetic differences between Chilean Map isolates from different geographic and host sources. Tracing with regard to these factors are epidemiologically important. The study described isolates of Map from different Chilean animal host species using IS1311. The typing result for the guanaco isolates indicates that these animals have not been infected from the sheep, more likely from cattle or goats. PCR-REA as a simple and rapid test that can be used on a range of diagnostic samples for the confirmation of paratuberculosis and will be of benefit in control and eradication programmes for this disease.