The extraction and characterization of DNA from aquatic environmental samples offers an alternative, non-invasive approach for the detection of rare species. Environmental DNA, coupled with PCR and next-generation sequencing (“metabarcoding”), has proven to be very sensitive for the detection of rare aquatic species. Our study used a custom designed group-specific primer set and next-generation sequencing for the detection of three species at risk; (Eastern Sand Darter, Ammocrypta pellucida; Northern Madtom, Noturus stigmosus; and Silver Shiner, Notropis photogenis), one invasive species (Round Goby, Neogobius melanostomus) and an additional 78 native species from two large Great Lakes tributary rivers in southern Ontario, Canada; the Grand River and the Sydenham River. Out of 82 fish species detected in both rivers using capture-based and eDNA methods, our eDNA method detected 86.2% and 72.0% of the fish species in the Grand River and the Sydenham River, respectively, which included our four target species. Our analyses also identified significant positive and negative species co-occurrence patterns between our target species and other identified species. Our results demonstrate that eDNA metabarcoding that targets the fish community as well as individual species of interest provides a better understanding of factors affecting the target species spatial distribution in an ecosystem than possible with only target species data. Additionally, eDNA is easily implemented as an initial survey tool, or alongside capture-based methods, for improved mapping of species distribution patterns.