We investigated the spatial variability of picocyanobacterial community structure across the Costa Rica Dome (CRD), an offshore upwelling system characterized by high seasonal abundance of Synechococcus spp. We constructed clone libraries of the rpoC1 gene to survey picocyanobacterial diversity and developed specific real‐time quantitative polymerase chain reaction assays to assess the distribution of genetically distinct Synechococcus (SYN) and Prochlorococcus (PRO) populations across vertical and horizontal physicochemical gradients. Flow cytometry data showed that cell abundances for both SYN and PRO were highest near the dome center. Phylogenetic analysis of rpoC1 sequences revealed a remarkably high and distinctive picocyanobacterial diversity (FLU1‐3, CRD1, Clade II, XV, XVI) that included “novel” SYN and PRO genotypes. Furthermore, genetically different populations exhibited vertical and horizontal spatial partitioning. Abundances of distinct SYN genotypes peaked at subsequent depth horizons, leading to a fine vertical structure with at least three populations stacked within the upper 30–40 m at the dome. Clade II and FLU1A peaked in surface waters, while maximum concentrations of CRD1, FLU1B, and Clade XVI occurred in the upper and lower thermocline, respectively. Horizontally, Clade II abundance in surface waters remained high across the entire region, while SYN genotypes CRD1 and FLU1A increased with shoaling of the thermo‐ and nutricline toward the center of the dome to become the dominant genotypes of the SYN assemblage in the dome. Below the mixed layer, Clade XVI and PRO genotype FLU2, virtually absent outside the dome, became abundant components of the picocyanobacterial assemblage. Despite their phylogenetic relatedness, FLU1A and FLU1B subclades followed different distributional patterns, suggesting ecological significance of the microdiversity within the clade. The unprecedented fine vertical structure demonstrated for SYN genotypes is driven by sharp physicochemical gradients (e.g., density, nutrient, oxygen, and trace metals) created by the dome and the presence of a shallow oxycline that enhances habitat diversification.