This USGS data release documents presence and absences of 419 fish species in the conterminous United States for 35,918 stream reaches of the National Hydrography Dataset Plus Version 2.1 (NHDPlusV2.1). Sample dates for this dataset span 1990-2019. Fish samples were aligned to the Integrated Taxonomic Information System (ITIS), where each species record was assigned a Taxonomic Serial Number (TSN). The dataset is structured with records representing a stream reach (i.e. comid), sample description (i.e. source, date) and a series of 419 columns representing species where each species column is named as the TSN. Data can be visualized on the NHDPlusV2.1 after a tabular join using the field 'comid' or can be projected and visualized as point data using the latitude and longitude fields (using coordinate reference system NAD83) that represent the midpoint of the stream reach that they were associated with. Data are provided in comma separated value (CSV) format.