We conducted a controlled feeding trial using two captive river otters Lontra canadensis to determine how prey size may introduce bias into frequency of occurrence analysis using otter scats. Otters were fed specific prey across a range of sizes. We then collected all scats deposited by the otters to determine how many defecation events occurred to remove the prey item from the digestive system. We found a strong, positive relationship between prey item size and the number of scats required to excrete the item. We then examined how the results, of an actual river otter feeding habits study using frequency of occurrence analysis of scats, could be biased towards an over-representation of larger prey items by using a correction factor for prey item size developed from our feeding trials. Frequency of occurrence suggested a strong preference for mid-range size of prey items and a strong avoidance of smaller prey items. Our corrected results indicated that otters exhibit little preferential feeding based on prey item size in the Missouri Ozarks. Our results suggest that bias associated with frequency of occurrence analyses may severely limit the robustness of inferences that can be made from such analyses.