AbstractDinoflagellates constitute an abundant and diversified component of marine plankton, mostly associated with stratified conditions typical of late spring through autumn in temperate regions. Yet, difficulties with the identification of many species limit the knowledge of their composition and seasonal succession. Here we use a 3‐year V4‐18S rDNA metabarcoding dataset (4,366,007 reads, 4650 Amplicon Sequence Variants, ASVs) collected on 48 dates at the Long‐Term Ecological Research site MareChiara (LTER–MC) to explore the diversity and temporal distribution of dinoflagellates in the Gulf of Naples (Mediterranean Sea). A large fraction of dinoflagellate ASVs (55%, 68% of the reads) were assigned to 187 species in 85 genera, while 33% (23% of the reads) were attributed to undetermined Syndiniales, and the remainder to unidentified groups. A total of 147 ASVs were assigned at 100% similarity to 135 reference sequences, corresponding to 116 species, 46 of which were putative new records for the Gulf of Naples. Cluster analysis conducted on a normalized dataset of 1199 ASVs identified four sample clusters that corresponded largely to the spring, summer, autumn and winter seasons, respectively, each including samples from all 3 years. Syndiniales were particularly abundant (43.1%) in the winter cluster, whereas Gyrodinium reached higher percentages in the summer and autumn ones (38.6% and 57.8%), Gymnodiniales sensu stricto were more abundant in spring (11.5%) and Gonyaulacales in summer (13.5%). Almost half of the ASVs analysed (561) were significantly associated with one of the four seasonal clusters (p < .05). The winter group of ASVs was particularly rich (348 ASVs) and mainly consisted of naked and parasitic taxa, which are impossible to identify in routine phytoplankton observations in light microscopy. Despite biases mainly ascribed to both the incompleteness of the reference dataset and the limited resolution of the marker gene, the metabarcoding approach has provided new insights into the ecology and distribution of this important component of the plankton, highlighting its marked seasonality along with the existence of a diversified and previously overlooked dinoflagellate community in winter.