Although Escherichia coli O157:H7 prevalence estimates in cattle have increased over time due to improvements in detection methods, fecal sample transport conditions from farm to microbiological laboratories for further analysis may be a factor for prevalence underestimation. The objective of this study was to compare and determine the survival characteristics of E. coli O157:H7 in bovine feces under various storage conditions that could be encountered during transport. Fecal pats were inoculated with a four-strain cocktail of antibiotic-resistant E. coli O157:H7 to contain ≃1 × 105 CFU/g. Inoculated and control samples were taken after 0, 24, 48, 120, and 168 h at each storage temperature and examined for presence and numbers of E. coli O157:H7. Each sample was subdivided and placed at each of the four following temperatures: 37, 23, 4.4°C, and in a plastic cooler with refrigerant packs (0, 4, 4, 21, and 23°C at five sampling times, respectively) to simulate transportation conditions. A statistically significant decrease in the population of the pathogen was observed after 48 h in samples held at 37°C (P < 0.01) and after 168 h at 4.4°C (P = 0.02). At 37°C, E. coli O157:H7 was not detected after 48 h, either by direct plating (P < 0.01) or by immunomagnetic separation. Overall, the results of this study showed that E. coli O157:H7 survived without significant detriment in bovine fecal material inside the cooler for up to 168 h. These results indicate that shipment and storage under these conditions before microbiological analysis would be acceptable and should not affect pathogen detection.