Environmental DNA (eDNA) is emerging as a potentially powerful tool to assess aquatic ecosystems, and to monitor fish assemblages. We conducted a pilot eDNA water sampling program at 5 Reserves within the National Estuarine Research Reserve System from temperate and tropical estuaries to better understand how eDNA data can support detection of fish species within an standardized monitoring network. Samples were collected in coordination with an existing long term monitoring program, and the sampling plan was designed to minimize additional work for field teams. We found that results varied; in some estuaries the number and type of fish species was consistent with expected occurrence, and eDNA analysis detected fish that often eluded traditional sampling methods. In warm turbid waters, however, we detected fewer species than expected, suggesting that additional processing is required at these sites. Managers interested in applying standardized eDNA monitoring across a range of sites may want to consider a decision tree protocol, where a baseline methodology is developed for all sites, enhanced by additional laboratory or analysis steps when specific conditions are present.