Technical Abstract: Clinical associations between Crohn’s disease in humans and Mycobacterium avium subspecies paratuberculosis (Map) have been suggested but not confirmed. Map is the causative agent for Johne’s disease in cattle. Infected cattle could be sources for Map transmission to humans via dairy and beef products, but no information on Map prevalence with beef or beef products has been reported. More than 2,200 samples of ileocecal lymph nodes and swabs of hides and carcasses from 341 animals at cull-cattle and 245 animals at fed-cattle slaughter facilities across the United States were analyzed for the presence of Map. Molecular analysis using amplification of the IS900 and/or ISMap02 genetic sequences detected Map DNA predominantly on hides and in lymph nodes of samples taken at both types of processing facilities. More than 34% of the cattle at cull cow slaughter facilities had ileocecal lymph nodes testing positive for Map DNA. From these same cattle, hide prevalence was more than 2-fold greater than prevalence in ileocecal lymph nodes, suggesting that cross-contamination could be occurring during transport and lairage. The prevalence of Map-specific DNA decreased during processing and less than 11% of the carcasses tested positive after interventions in the cull cow processing facilities. Using standard double decontamination and culture techniques, less than 1% of the post-intervention carcasses tested positive for viable Map at cull cow facilities. In samples from the facilities processing only fed cattle, Map prevalence of 1% or less was detected for ileocecal lymph node, or hide and carcass samples, and viable Map were not detected on fed beef carcasses. Based on this study, fed-cattle carcasses are unlikely sources of Map and carcasses at cull cow plants have only a slight risk for transmitting viable Map due to the effectiveness of the antimicrobial interventions during processing.