Abstract Environmental DNA metabarcoding has become a popular tool for the assessment of freshwater biodiversity, but it is largely unclear how sampling time and location influence the assessment of communities. Abiotic factors in rivers can change on small spatial and temporal scale and might greatly influence eDNA metabarcoding results. In this study, we sampled three German rivers at four locations per sampling site: 1. Left river side, surface water 2. Right river side, surface water, 3. Left side, close to the riverbed, 4. Right side, close to the riverbed. For the rivers Ruhr and Möhne, sampling was conducted three times in spring, each sampling one week apart. The Ruhr was again sampled in autumn and the Gillbach was sampled in winter. Sequencing on an Illumina MiSeq with COI primers Bf2/BR2 revealed diverse communities (6493 Operational taxonomic units, OTUs), which largely differed between rivers. Communities changed significantly over time in the Ruhr, but not in the Möhne. Sampling location influenced recovered communities in the Möhne and in the Ruhr in autumn. Our results have important implications for future eDNA studies, which should take into account that not all eDNA in rivers is everywhere and not at all times.