The eastern tropical Pacific Ocean holds two of the main oceanic oxygen minimum zones of the global ocean. The presence of an oxygen-depleted layer at intermediate depths, which also impinges on the seafloor and in some cases the euphotic zone, plays a significant role in structuring both pelagic and benthic communities, and also in the vertical partitioning of microbial assemblages. Here, we assessed the genetic diversity and distribution of natural populations of the cyanobacteria Prochlorococcus and Synechococcus within oxic and suboxic waters of the eastern tropical Pacific using cloning and sequencing, and terminal restriction fragment length polymorphism (T-RFLP) analyses applied to the 16S-23S rRNA internal transcribed spacer region. With the T-RFLP approach we could discriminate 19 cyanobacterial clades, of which 18 were present in the study region. Synechococcus was more abundant in the surface oxic waters of the eastern South Pacific, while Prochlorococcus dominated the subsurface low-oxygen waters. Two of the dominant clades in the oxygen-deficient waters belong to novel and yet uncultivated lineages of low-light adapted Prochlorococcus.