AbstractPicophytoplankton are important primary producers, but not always adequately recognized, for example, due to methodological limitations. In this study, we combined flow cytometry and metabarcoding to investigate seasonal and spatial patterns of picophytoplankton abundance and community composition in the Elbe estuary. Due to the mixing of freshwater and seawater and the tidal currents this ecosystem is characterized by typical estuarine features such as salinity gradients and high turbidity. Picophytoplankton (mostly picoeukaryotes such as Mychonastes and Minidiscus) contributed on average 70% (SD = 14%) to the total phytoplankton counts. In summer picocyanobacteria (e.g., Synechococcus) played a more significant role. The contributions of picophytoplankton to the total phytoplankton were particularly high from summer to winter as well as in the mid estuary. However, at salinities of around 10 PSU in the mixing area of freshwater and seawater, the proportion of picophytoplankton was comparably low (average 49%, SD = 13%). Our results indicate that picophytoplankton prevail in the Elbe estuary year‐round with respect to cell counts. Picophytoplankton could occupy important niche positions to maintain primary production under extreme conditions where larger phytoplankton might struggle (e.g., at high or low temperature, high turbidity, and in areas with high grazing pressure) and also benefit from high nutrient availability here. However, we did not find evidence that they played a particularly significant role at the salinity interface. Our study highlights the importance of including picophytoplankton when assessing estuarine phytoplankton as has been suggested for other ecosystems such as oceans.