The aim of this study was to search for Mycobacterium avium Subsp. paratuberculosis (Map) infection in a free-ranging wild animal species in a region where Johne's disease has yet to be reported and to classify Map isolates using a genomic typing method. Fecal samples were obtained from 501 wild guanacos (Lama guanicoe) from Tierrra del Fuego Island, Chile, in August 2006. Samples were cultured using Herrold's egg yolk medium with mid without mycobactin J. After 9 mo of incubation, suspected Map colonies showing mycobactin dependence were confirmed by real-time polymerase chain reaction (PCR) based on IS900 and F57. Isolates were further tested using IS1311 PCR with restriction endonuclease analysis in order to type the guanaco Map strains. Twenty-one of 501 (4.2%) animals were fecal culture-positive for Map; identity was confirmed by real-time PCR and isolates were classified as cattle-type. Most culture- positive animals were located in four contiguous geographic areas, and the infection was most commonly found among adult animals. Prevalence was higher in females (5.9%) than males (3.1%) but the difference was not statistically significant. This represents the first isolation of Map from a free-ranging wildlife species in Chile. It expands the geographic range of paratuberculosis and the diversity of wildlife species that call become infected with Map.