Non-wadeable river systems are one of the most diverse aquatic ecosystems in Iowa; however, fish assemblages in these systems are virtually unknown. One of the primary reasons little is known about fishes in non-wadeable rivers is because few studies have investigated methods for adequately sampling fishes in these systems. Therefore the purpose of this study was to develop a sampling protocol for fishes in non-wadeable rivers in Iowa. In 2007 and 2008, 21 sites were sampled on 16 non-wadeable rivers throughout the state of Iowa. Sites were three or five kilometers in length depending on stream order. Each site was sampled with boat-mounted electrofishing equipment, a modified Missouri trawl, and a bag seine. A total of 21,292 fish from 84 species in 17 families were collected including 23 species of greatest conservation need and one state threatened species. The results of this sampling were then used to determine appropriate gears and estimate the number of samples required to observe various percentages of the total number of species observed at each site. Electrofishing sampled the greatest number of species and the greatest number of species not sampled by the other gears. Trawling sampled the highest number of species of greatest conservation need (SGCN), while seining sampled the fewest species and SGCN. As a management recommendation, we recommend 2,500 m of electrofishing combined with 42 50-m long trawls, which was the mean, minimum effort necessary to sample 90% of the species observed with a 95 probability of detection. Additionally, sample reaches could be categorized into one of three major groups based on their fish assemblage. Groups were based predominantly on stream geomorphology (i.e., depth, width, substrate, and drainage basin).