Phospholipid (PL) composition has a tremendous influence on the cell integrity and physiological competency. At the same time, plankton PL make important metabolic fuels for higher trophic levels. The goal of this study was to identify environmental control on PL production and their molecular identity of the suspended particles in two different estuaries. We conducted research in subtropical, eutrophic Wenchang River Estuary in China and temperate pristine, mesotrophic Krka River Estuary in Croatia. In agreement with the more abundant phytoplankton, PL concentrations were much higher in the Wenchang River Estuary (30.3–178.2 μg L⁻¹) than in the Krka River Estuary (8.4–18.8 μg L⁻¹). Given that six PL classes investigated (phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), and phosphatidylserine (PS)) have different roles in the cell, we expected their different fatty acid composition in different environments. We found small differences in the fatty acid composition of PC, PG, and PI between two estuaries. These results suggest that the essential fatty acid compositions of these PL in estuarine plankton are relatively constant in order to preserve membrane functions and/or cell processes in which they are involved regardless of environmental conditions. In contrast, PE, PA and PS fatty acid composition substantially differed between two estuaries as well as throughout the salinity gradient in each estuary. This suggests the adaptability of plankton to remodel these PL depending on the environmental conditions and the plankton community structure. Good environmental conditions (favorable N/P ratio, temperature) are important for increased PL content (% in POC and total lipids) in estuarine plankton and increased essential polyunsaturated fatty acid content in PL, which is beneficial to higher trophic levels.