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Estuaries are highly productive ecosystems, which are strongly affected by several anthropogenic pressures. Phytoplankton is a key element for assessing the ecological quality status in these transitional waters. Moreover, understanding physico-chemical and biological drivers is crucial to disentangle their effect on the structure of phytoplankton community. The present work aims to study the effect of the main physico-chemical drivers on the phytoplankton community structure and dynamics in a temperate well-mixed estuary (Sado Estuary). Four sampling stations were analyzed monthly in three regions of the estuary, from 2018 to 2019. Surface water samples were collected to analyze the phytoplankton community and several concomitant physico-chemical parameters. Temperature, turbidity, salinity, and nutrients availability were the drivers that best explained the spatio-temporal patterns observed in the phytoplankton community. The upper estuary was characterized by higher phytoplankton cell abundances and biomass. Three phytoplankton groups stood out in the characterization of the estuarine assemblages: diatoms, cryptophytes, and dinoflagellates. Diatoms were the dominant group most of the year, being dominated by small cell species (single and chain-forming) upstream, and by larger chain-forming species downstream. Cryptophytes had a high contribution to the community in the inner regions of the estuary, while dinoflagellates contributed more for the community composition downstream, where high abundances of harmful algal species were sporadically found. Previous studies on the phytoplankton community dynamics in this estuary are limited to the 1990s. Thus, the present study provides insight into changes in the dominant phytoplankton groups of the Sado Estuary in the last 25 years, namely an increase in cryptophytes over diatoms in the inner estuarine regions, and an increase in dinoflagellates near the estuary mouth., (© 2022. The Author(s).)