Environmental DNA (eDNA) samples were taken across the Thames catchment during 2018. Water samples were filtered from the freshwater and tidal Thames using Sterivex capsule filters, and amplified with fish specific primers targeting the 12S (Miya et al., 2015) and CO1 (Shokralla et al., 2015) regions, before Illumina MiSeq sequencing. The resultant FASTA files and collection meta data are included here.