Significance Metagenomics has become an accessible approach to study complex microbial communities thanks to the advent of high-throughput sequencing technologies. However, molecular ecology studies often face interpretation issues, notably due to the lack of reliable reference databases for assigning reads to the correct taxa and use of fixed cutoffs to delineate taxonomic groups. Here, we considerably refined the phylogeography of marine picocyanobacteria, responsible for about 25% of global marine productivity, by recruiting reads targeting a high-resolution marker from Tara Oceans metagenomes. By clustering lineages based on their distribution patterns, we showed that there is significant diversity at a finer resolution than the currently defined “ecotypes,” a diversity that is tightly controlled by environmental cues. , Prochlorococcus and Synechococcus are the two most abundant and widespread phytoplankton in the global ocean. To better understand the factors controlling their biogeography, a reference database of the high-resolution taxonomic marker p et B , encoding cytochrome b 6 , was used to recruit reads out of 109 metagenomes from the Tara Oceans expedition. An unsuspected novel genetic diversity was unveiled within both genera, even for the most abundant and well-characterized clades, and 136 divergent petB sequences were successfully assembled from metagenomic reads, significantly enriching the reference database. We then defined Ecologically Significant Taxonomic Units (ESTUs)—that is, organisms belonging to the same clade and occupying a common oceanic niche. Three major ESTU assemblages were identified along the cruise transect for Prochlorococcus and eight for Synechococcus . Although Prochlorococcus HLIIIA and HLIVA ESTUs codominated in iron-depleted areas of the Pacific Ocean, CRD1 and the yet-to-be cultured EnvB were the prevalent Synechococcus clades in this area, with three different CRD1 and EnvB ESTUs occupying distinct ecological niches with regard to iron availability and temperature. Sharp community shifts were also observed over short geographic distances—for example, around the Marquesas Islands or between southern Indian and Atlantic Oceans—pointing to a tight correlation between ESTU assemblages and specific physico-chemical parameters. Together, this study demonstrates that there is a previously overlooked, ecologically meaningful, fine-scale diversity within some currently defined picocyanobacterial ecotypes, bringing novel insights into the ecology, diversity, and biology of the two most abundant phototrophs on Earth.