Technical Abstract: Clostridium difficile-associated disease has recently increased in both illness and relapse rates in North American and European countries. This increase has been attributed to the emergence of a toxigenic strain designated as North America pulsed-field gel electrophoresis type 1 or NAP-1. The NAP-1 strain has been isolated from calves’ feces, as well as retail ground beef in Canada, leading to a speculation of illnesses from consumption of contaminated meat products. However, information on C. difficile associated with U.S. beef cattle during processing and in commercially produced ground beef is limited. In order to determine this missing information, samples from various steps during beef production were collected. Each of 525 hides, pre-evisceration, and post-intervention carcasses, and 1,048 commercial ground beef samples were collected across the United States. No C. difficile was detected on pre-evisceration and post-intervention carcasses, or in commercially produced ground beef. The prevalence of C. difficile on hides was 3.2%. A total of 18 isolates from hides were phenotypically and genetically characterized. None of the isolates was identified as NAP-1 strain. The results of this study establish a baseline for prevalence of C. difficile in the U.S. beef processing industry, which has a low risk associated with this pathogen.