This dataset contains raw sequence data collected from an eDNA metabarcoding project to detect freshwater mussel species across two sites in Georgia (Spring Creek and Flint River) and one drainage in Missouri (Big Piney River). The eDNA samples were collected from each stream using dead-end ultra filtration (D-HFUF) with eDNA extracted from filters. We used two previously published primer sets designed to amplify freshwater mussels, one that amplified the mitochondrial cytochrome c oxidase I (COI) region and one that amplified the NADH dehydrogenase subunit I region, with negative control and mock community samples included. Samples were individually indexed, pooled, and sequenced on an Illumina MiSeq high-throughput sequencer. Raw reads were processed with bioinformatic scripts to identify unique sequences and filter out low-quality reads. Reads were assigned to mussel species using the NCBI executable BLAST v2.12.0+ and the NCBI nt database.