An eDNA-based sampling approach used by the U.S. Fish and Wildlife Service for bigheaded carp Hypophthalmichthys spp. in the upper Mississippi River and Great Lakes Basins that collects hundreds of water samples per event. The U.S. Fish and Wildlife Service detected a single positive sample for bighead carp H. nobilis during the spring 2021 sampling event in the Kinnickinnic River within the Milwaukee River Basin, and detected a second single positive sample for bigheaded carp during the fall 2021 sampling event in the Milwaukee River. The U.S. Fish and Wildlife Service did not detect any bigheaded carp or bighead carp in previous years (2015 to 2020) or in either the spring or fall 2022 sampling events. These detections lacked perspective such as detection numbers for other species. We reanalyzed the 2021 and 2022 samples for four existing species of fish: two fairly common species (common carp Cyprinus carpio and gizzard shad Dorosoma cepedianum) and two fairly rare species (burbot Lota lota and grass carp Ctenopharyngodon idella). This dataset contains the eDNA-base detection and nondetection data from the 2021 and 2022 sampling events.