Diatoms are one of the most important phytoplankton groups in the world's oceans. They are responsible for up to 40% of the photosynthetic activity in the Ocean, and they play an important role in the silicon and carbon cycles by decoupling carbon from atmospheric interactions through sinking and export. These processes are strongly influenced by the taxonomic composition of diatom assemblages. Next-generation sequencing enabled us to study diversity deeper than before and uncover new distribution patterns. However, phylogenetic markers used for this purpose, such as various 18S rDNA regions, are often not powerful enough to distinguish certain taxa. Here, we compared the chloroplast-encoded rbcL marker for metabarcoding marine diatoms to microscopy and 18S-V9 metabarcoding using a series of monthly samples from the Gulf of Trieste (GoT) in the northern Adriatic Sea. We demonstrate that rbcL is able to detect more taxa compared to 18S-V9 or microscopy, while the overall structure of the diatom assemblage was comparable to the other two methods with some variations, that were taxon dependent. In total, 6 new genera and 22 new diatom species for the study region were identified. Furthermore, on the example of two well-studied genera in the region, Chaeteceros and Pseudo-nitzschia, we show how the rbcL method can be used to infer even deeper phylogenetic and ecologically significant differences at the species population level. Our work has implications for studies dealing with taxa distribution and population structure, as well as carbon and silica flux models and networks.