The aim of presented researches was to investigate the physicochemical properties of Langmuir monolayer of galactolipids extracted from two different kinds of plastids: immature embryos and inflorescences. Differences between the physicochemical properties of the plastid membranes may help to explain different physiological processes, such as plant regeneration. Surface pressure (π) vs. molecular area (A) isotherms of the monogalactosyldiacylglycerol (MGDG)/digalactosyldiacylglycerol (DGDG) monolayers of various molar ratios were measured at 15°C. Galactolipids were extracted from two different types of tissue: inflorescences and embryos. Based on the analysis of the π–A isotherms, the properties of monolayers, such as collapse pressure (π coll ), limiting area (A lim ), compressibility modulus (Cs−1), excess free energy of mixing (ΔG EXC ) and free energy of mixing (ΔG MIX ), were calculated. The results show that pure MGDG and DGDG and their mixtures form liquid-expanded monolayers, independently on the kind of tissue. Galactolipids originating from inflorescences produce more compressible films at the air/water interface, with larger limiting area per molecule and lower stability against the collapse process. MGDG and DGDG are miscible and form non-ideal mixed monolayers at the air/water interface. Negative values of ΔG EXC were calculated for the mixture of galactolipids originating from inflorescences, with the content of MGDG, x MGDG >0.6. In the case of embryos, the negative values of ΔG EXC were found for x MGDG ≈0.5. Therefore, the attractive interactions between MGDG and DGDG exist in the mixtures of these compositions. As it is shown by negative values of ΔG MIX , mixed monolayers are more stable compared with unmixed ones.