Johne's disease, a fatal and contagious gastrointestinal infection caused by Mycobacterium avium subsp. paratuberculosis (Map), was first diagnosed in an endangered Florida Key deer (Odocoileus virginianus clavium) in 1996 and later in six additional Key deer deaths from 1998 to 2004. We investigated the geographic distribution of Map in the Lower Florida Keys from February 2005 through May 2006 via collection of blood and fecal pellets from 51 live-captured deer, collection of 550 fecal samples from the ground, and by necropsies of 90 carcasses. Tissue and fecal samples also were submitted from 30 raccoons (Procyon lotor), three feral cats (Felis catus), an opossum (Didelphis virginiana), and a Lower Keys marsh rabbit (Sylvilagus palustris hefneri). Mycobacterium avium subsp. paratuberculosis was identified in 23 Key deer fecal samples collected from the ground, tissue samples from two clinically ill Key deer, and from the mesenteric lymph node of a raccoon. The results of this study indicate that Map persists in the Key deer population and environment at a low prevalence, but its distribution currently is limited to a relatively small geographic area within the range of Key deer.