Technical Abstract: Background. Escherichia coli O157:H7 contamination of beef is an important food safety concern. Cattle are a natural reservoir of this pathogen, and the life-cycle of E. coli O157:H7 involves cycling between the warm, nutrient-rich cattle large intestine and the generally cool, nutrient-limiting feedlot pen external to the animal. Despite the important role that the feedlot pen may play in the transmission of E. coli O157:H7 among animals, and its potential contribution to E. coli O157:H7 contamination of the final food product, little is understood about the microbial ecology of beef cattle feedlot pens, which are commonly conceptualized as being packed uncomposted manure. We have therefore undertaken a study to characterize the bacterial communities of the beef cattle feedlot pen surface material using 3,647 near full-length 16S rDNA sequences. Results. The feedlot surface composite was well represented by members of the phylum Actinobacteria (42%), followed by Firmicutes (24%), Bacteroidetes (24%), and Proteobacteria (9%). The feedlot pen surface material bacterial communities were clearly distinct from those of the feces from animals in the same pen. Comparisons with previously published results of feces from the animals in the same pen reveal that, of 139 genera identified, only 25 were present in both habitats. Conclusions. These results indicate that the feedlot pen surface material is more than “just manure”, and strongly suggest that E. coli O157:H7 may face different selection pressures, and therefore be vulnerable to different control measures during its tenure in the feedlot pen, as compared to its residence in the bovine gastrointestinal tract.