Environmental DNA (eDNA) is DNA that has been released by an organism into its environment, such that the DNA can be found in air, water, or soil. In aquatic systems, eDNA has been shown to provide a sampling approach that is more sensitive for detecting target organisms faster, and less expensively than previous approaches. However, eDNA needs to be sampled in a manner that has been tested and found effective and, because single copies of target DNA are detected reliably, rigorous procedures must be designed to avoid sample contamination. Here we provide the details of a sampling protocol designed for detecting fish. This protocol, or very similar prototypes, has been used to collect data reported in multiple peer-reviewed journal articles and from more than 5,000 additional samples at the time of publication. This process has been shown to be exceedingly sensitive and no case of field contamination has been detected. Over time, we have refined the process to make it more convenient. Our policy at the National Genomics Center for Wildlife and Fish Conservation is to provide collaborators with kits that contain all of the materials necessary to properly collect and store eDNA samples. Although the instructions in this protocol assume that the collaborator will have this same equipment, we also describe how users can create their own kit, and where we think there is flexibility in the equipment used.