Mycobacterium avium subsp. paratuberculosis(MAP) is the causative agent of paratuberculosis. The primarily affected hosts are domestic ruminants, but paratuberculosis has also been reported in wild animal hosts. In Chile, the infection has been confirmed in cattle, sheep and goats, but there is no information about paratuberculosis in wildlife animals. The broad range of hosts affected by MAP implies a possible intraspecies transmission, as well as wildlife reservoir. Guanaco is the only wild ungulate species widely distributed across the Patagonian steppe, sharing grazing land with domestic sheep. The aim of this study was to detect MAP infection in a free ranging wildlife animal species in Chile, using conventional diagnostic tools, as well as new molecular confirmation technology. Faecal samples were obtained from 501 guanacos populating the Rusffin area, Timmaukel County in Tierra del Fuego Island in August 2006. The sampling was synchronized with a controlled hunting activity carried out by a private company under the Ministry of Agricultural (SAG) supervision. Faecal samples were collected post mortem right after hunting and cultured on a homemade HEY medium with and without mycobactin J following the procedure recommended by the Cornell University. Colonies resembling MAP and showing mycobactin-dependence were confirmed by Real -Time PCR based on IS900 and F57. Twenty one out of 501 (4.2%) animals sampled were positive for Map all of which were confirmed by Real -Time PCR IS900 and F57. This represents the first isolation of Map from a free-ranging wildlife animal in Chile. These findings support an increasing body of evidence that indicates that a wide diversity of wildlife species as well as domestic ruminants can become infected with Map. In a control or eradication program of this disease, it is of special importance to know how to control the transmission. The presence of a wildlife reservoir of the disease has to be considered for the potential transmission to livestock. However, for free-ranging wildlife, the most likely initial source of infection is the shared range with domestic species, given the higher prevalence of Johne's disease in the latter species, an issue to be determined in the Chilean situation.