Understanding the origin and maintenance of genetic diversity is crucial to elucidate population dynamics of unicellular microalgae, their microevolutionary history and their adaptive ability. The planktonic, domoic acid-producing diatom Pseudo-nitzschia multistriata has a ubiquitous distribution in the world oceans and past population genetics studies, based on few genomic loci, have shown a clear temporal structure over different years in the Gulf of Naples (Italy). Despite the ecological and toxicological importance of this organism, detailed information on its diversity across the whole genome and at the population level is still lacking. We collected P. multistriata strains in the Gulf of Naples in five different years, obtained strains from the Adriatic Sea, the Gulf of Mexico and New Zealand coasts, and resequenced the whole genomes of a total of 28 strains at high coverage. While strains from the first three geographical areas were capable of producing the toxin domoic acid, the New Zealand strains had been reported to be non-toxic. A comparison of the domoic acid biosynthetic (dab) genes sequences between toxic and non-toxic strains showed very little variation among the strains, and no disrupting mutation was found in the dab genes in the non-toxic strains. On the other hand, the dab genes showed higher levels of expression in toxic strains than in non-toxic strains, suggesting that, in this species, absence of toxicity is explained by gene regulation rather than dab sequence divergence. Variant analysis showed stronger spatial than temporal genetic structuring and a clear separation was observed between the New Zealand strains and the others, the former having a greater content of genes under selection. Overall, the genomes of the different groups, including strains from a clonal bloom, did not appear to contain major rearrangements. Our findings contribute to enlarging our understanding of diatom diversity, a key factor underlying diatom success, and provide novel data on the longstanding problem of Pseudo-nitzschia toxicity.