To evaluate site-to-site variation within fecal pats from cattle with regard to detection of Escherichia coli O157 and determine the effect on the accuracy of prevalence estimates of assay of multiple samples collected from the same fecal pat. 120 freshly voided fecal pats collected from 2 beef feedlots. Procedures-5 samples were systematically collected from each fecal pat and analyzed for E coli O157 via selective preenrichment techniques, immunomagnetic separation, and biochemical tests. Presumptive isolates were definitively identified via agglutination assays and polymerase chain reaction techniques. Best estimators of prevalence were calculated from the distribution of E coli O157-positive samples per pat. Of the 120 fecal pats, 96, 13, 4, 2, 3, and 2 fecal pats had 0, 1, 2, 3, 4, and 5 E coli O157-positive samples, respectively. The greatest estimate of E coli O157 prevalence (20%) was achieved when all 5 samples were assessed; this estimate represented a 2.4- fold increase in prevalence, compared with that provided via analysis of 1 sample/pat (8.2%). Compared with assessment of 5 sites/pat, the relative sensitivity of detecting an E coli O157-positive fecal pat via analysis of 1 site/pat was 40.1%. Results suggest that estimates of E coli O157 prevalence derived from sampling of 1 location/pat are likely underestimates of the true prevalence of this pathogen in fecal pats (and by extension, cattle). Additional research is warranted to confirm these results in situations of high and low prevalence and across different feedlots.