AbstractDiatoms’ bloom dynamics, with seasonal cycling of waxes and wanes of cell abundance, imply frequent bottleneck and expansion events that can leave signatures in the genetic structure and diversity of populations. We explored changes in genetic diversity and structure within multiple species of the planktonic marine diatom genus Pseudo‐nitzschia living in sympatry in the Gulf of Naples (Tyrrhenian Sea, Italy) over 48 sampling dates (from 2011 to 2013) by means of metabarcode data. The genus Pseudo‐nitzschia includes complexes of cryptic species along with morphologically distinguishable ones. We assessed the resolution power of the hypervariable V4 region of the 18S rDNA in delimiting species using different algorithms and a set of reference sequences. We measured intra‐specific genetic diversity and differentiation among species over three annual cycles. Between‐species divergence was higher in the P. pseudodelicatissima complex, while signs of incomplete lineage sorting and/or introgression were detected in the P. delicatissima complex. Most of the species showed significant population differentiation over time, due to changes in haplotypic composition over the three years. Some haplotypes were lost and new ones appeared, with high polymorphism in species of the P. delicatissima complex. We hypothesize that the observed temporal changes in genetic structure result from frequent founder events linked to a population dynamic characterized by cyclical alternation of clonal expansions and bottlenecks and discuss possible factors affecting the microevolutionary pathways in these planktonic diatoms.