Much of our current knowledge onProchlorococcushas derived from research on various genetic strains that have successfully been brought into culture. In particular, analyses of the complete genomes of 12 of those isolates have revealed the extent to which these strains differ from each other and the genetic means by which they are adapted to specific environmental niches. However, based on culture-independent studies it is now clear that the strains currently available in diverse culture collections do not represent the true diversity ofProchlorococcusgeno- and phenotypes. Potential alternatives to overcome the limitations caused by difficulties in isolatingProchlorococcusmay be provided by the whole-genome amplification of flow cytometrically isolated individual cells. The new information obtained in this way on various genetic types would, in turn, facilitate the correct identification ofProchlorococcus-derived sequences within metagenomic sequence data sets. However, culture-independent molecular population genetic approaches have also greatly furthered our understanding of the ecology and physiological capability ofProchlorococcusgenetic groups. Based on this, I support the notion that such population genetic approaches to reveal theProchlorococcusmicrodiversity are still of great value if appropriate marker genes providing a high genetic resolution are used. The comparison of the results from those culture-independent analyses of environmental marine samples with those from the measurement of relevant environmental and biotic parameters still has potential to uncover further parameters that controlProchlorococcusdiversity and distribution. In this context, I will discuss those markers that have so far been used in population genetic studies ofProchlorococcus, which is followed by outlining a general approach to evaluate their use for resolving microbial microdiversity and for phylogenetic identification.