Life in estuaries, especially in surface waters, is a challenge, particularly due to changes in salinity. Environmental changes inevitably lead to acclimation or adaptation of phytoplankton in order to survive. Since membranes are the first to perceive changes in the environment, we focused on understanding how phytoplankton in estuaries adapt to salinity stress through lipid remodeling. Since photosynthesis is one of the most sensitive processes, we studied the response of phytoplankton thylakoid membrane lipids to salinity stress. The study was conducted in two estuaries with completely different environmental characteristics. Apart from hydrology, estuaries also differ in phytoplankton community compositions, nutrient status and temperature. Here we show that estuarine phytoplankton, regardless of environmental differences in the two estuaries studied, increase monogalactosyldiacylglycerol (MGDG) content and unsaturation in response to osmotic shock to protect photosynthetic machinery. This was particularly pronounced at the lowest salinities when freshwater phytoplankton encounter saline water and decreases with increasing salinity. Our results also suggest that increased concentrations of nitrogen nutrients have a positive effect on the increased unsaturation of MGDG. Finally, we speculate that the freshwater green algae are the major group responsible for the observed largest increased and content of unsaturated MGDG at the lowest salinity.