Until recently, the evolutionary relationships between marine and freshwater microbes were unclear, but the use of molecular phylogenies is beginning to shed light on this subject. An increasing amount of studies are showing that marine and freshwater microbes (including viruses) are usually not closely related, often grouping into distinct marine and freshwater phylogenetic clusters, similar to what has been reported before for macroorganisms. These studies indicate that marine-freshwater transitions have been infrequent events during the diversification of microbes and that most of these transitions occurred a long time ago in evolutionary terms. Here we discuss the significance of recent studies addressing this question and consider possible avenues for future research.