To elucidate the degree of microdiversity within the genus Prochlorococcus, novel Prochlorococcus-specific polymerase chain reaction (PCR) primers were developed for the rpoC1 gene, which encodes the ribonucleic acid (RNA) polymerase core subunit. The size of the PCR fragment (925 bp) coupled with high sequence variation within the rpoC1 fragments (70-99% sequence similarity, 16S ribosomal RNA sequences show greater than 97% sequence similarity) meant that it was possible to distinguish Prochlorococcus strains by restriction fragment length polymorphism (RFLP) analysis. Clone libraries were constructed from environmental deoxyribonucleic acid samples from two stations, one in the northern and one in the southern oligotrophic gyre of the Atlantic Ocean. These were screened to determine the microdiversity of Prochlorococcus populations using this high-resolution high-throughput analysis approach. RFLP analysis of the clone libraries from the two gyre sites revealed that the two Prochlorococcus populations had a high degree of microdiversity with 40 and 52 different RFLP-type clones among the 143 clones tested for both the northern and southern gyres, respectively. Phylogenetic analysis of the nucleotide sequences of the RFLP types not only showed that it contained representatives of each of the currently recognized Prochlorococcus clades (based on the internal transcribed spacer region as molecular marker) but also led to the discovery of a previously unseen genetic microdiversity. This level of diversity was greater at the southern gyre site compared to the northern gyre site. Moreover, the high genetic resolution approach also revealed that there are two putative novel lineages within the HL I clade. Analyses of further samples by producing clone libraries from different geographic origins is likely to reveal further diversity and novel lineages within Prochlorococcus.