The presence of Mycobacterium avium subsp. paratuberculosis (MAP) in non-ruminant wildlife has raised questions regarding the role of these species in Johne's disease transmission. In this study we tested 472 tissues from 212 animals of six different species of scavenging mammals. All animals were taken from within a 210-square-mile area in Dane and Iowa counties of south central Wisconsin from September to May in 2003–04 and tested for the presence of MAP. We detected MAP-specific DNA in 81 of 212 (38%) scavenging mammals, in 98 of the 472 (21%) tissues; viable MAP was cultured from one coyote's ileum and lymph node tissue. Despite the low numbers of viable MAP isolated in this study, our data adds to the increasing evidence demonstrating the potential for transmission and infection of MAP in nonruminant species and provides possible evidence of interspecies transmission. The apparently high exposure of nonruminant wildlife provides potential evidence of a spill-over of MAP to wildlife species and raises the question of spillback to domestic and wild ruminants. These results demonstrate the importance of understanding the role of wildlife species in developing management strategies for Johne's disease in domestic livestock.