We investigated the community structure of Synechococcus and Prochlorococcus along a transect in the Arabian Sea during September 2001. The transect spanned contrasting oceanic conditions, allowing investigation of the effects of both horizontal and vertical environmental gradients over relatively large spatial scales on picocyanobacterial population structure. We applied previously developed oligonucleotide probes specific for different Prochlorococcus ecotypes and Synechococcus clades by hybridization to ‘oxygenic phototroph’ 16S ribosomal DNA polymerase chain reaction amplicons. Flow cytometry data showed that, in general, the picocyanobacterial community was dominated by Prochlorococcus in the southern oligotrophic waters and by Synechococcus in the northern mesotrophic waters. Molecular analysis of these picophytoplankton communities, however, revealed more specific spatial separation of lineages along the transect, with Prochlorococcus in southern surface waters being dominated by the high light‐adapted ecotype, while low light‐adapted (LL) ecotypes were confined to deeper waters below the surface‐mixed layer. Interestingly, between Sta. 2 to 4, the LL genotype MIT9303 appeared to be partitioned at the very base of the euphotic zone, beneath other LL genotypes. Most of the central and northern parts of the transect were dominated by Synechococcus genotypes of the clade II lineage. A significant exception was in the mesotrophic upwelling region, where genotypes representative of clades V/VI/ VII dominated. Members of the recently discovered clades IX and X were found in subsurface samples in warm, coastal waters. We propose that discrete differences, both horizontally and vertically, in a suite of environmental parameters along the transect provide optimal growth conditions for specific genotypes in a particular patch of water, giving rise to distinct spatial compartmentalization of picocyanobacterial lineages.