Determination of Escherichia coli O157 prevalence immediately prior to shipment and harvest is an important facet of the ecology of this organism, which requires further elucidation. As part of a larger study to measure the effects of within-pen prevalence of E. coli O157 on subsequent carcass contamination, fecal samples from 15 pens of cattle in each of 12 different feedlots in three states (Colorado, Nebraska, and Montana) were collected from June through September 2002. Thirty fresh fecal samples were collected from each pen floor within 36 h of shipment to slaughter. Fecal samples underwent standard enrichment, immunomagnetic separation, and isolation procedures for E. coli O157. Multivariable logistic regression was used to determine which factors best predicted pen-level positive culture results, and to estimate the magnitude of association between each factor and the outcome, while adjusting for other factors in the model. Thirteen (86.7%) of the 15 pens had at least one positive sample, and the within-pen prevalence of E. coli O157 in positive pens ranged from 3.3% to 77.8%. The odds of E. coli O157 positive fecal samples from cattle fed brewers grains were six times that for cattle not fed brewers grains. The odds of E. coli O157 positive fecal samples from pens of cattle from Central Nebraska was nine times that for pens of cattle from Eastern Colorado. These data demonstrate that the presence of E. coli O157 in fecal samples from finished feedlot cattle is associated with feeding of brewers grain and geographic location. Additional studies to further characterize geographic distribution of E. coli O157 and to investigate pen-level intervention strategies should be conducted.