Abstract: Tiny unicellular cyanobacteria or picocyanobacteria (0.5–3 µm) are important due to their ecological significance. Chesapeake Bay is a temperate estuary that contains abundant and diverse picocyanobacteria. Studies of Chesapeake Bay picocyanobacteria in the past 20 years led to the finding of new members of subcluster 5.2 Synechococcus. They laid the foundation for revealing the ecophysiology, biogeography, genomics, and molecular evolution of picocyanobacterial in the Chesapeake Bay and other coastal estuaries. The Bay picocyanobacteria are known to better tolerate the changes in temperature, salinity, and heavy metals compared to their coastal and open-ocean counterparts. Many picocyanobacteria isolated from the Bay contain rich toxin–antitoxin (TA) genes, suggesting that the TA system may provide them with a genetic advance to cope with variable estuarine environments. Distinct winter and summer picocyanobacteria are present in the Bay, suggesting a dynamic seasonal shift of the picocyanobacterial community in the temperate estuary. While the Bay contains subcluster 5.2 Synechococcus, it also contains freshwater Synechococcus, Cyanobium, and marine Synechococcus due to river influx and the ocean's tidal influence. Some Chesapeake Bay picocyanobacterial clades were found in the Bering Sea and Chukchi Sea, showing a link between the Bay and polar picocyanobacteria. Genomic sequences of estuarine picocyanobacteria provide new insight into the taxonomy and evolution of freshwater, estuarine, and marine unicellular cyanobacteria. Estuaries connect freshwater and marine ecosystems. This overview attempts to extend what we learned from Chesapeake Bay picocyanobacteria to picocyanobacteria in freshwater and marine waters.