The attractiveness of new omega-3 (ω-3) polyunsaturated fatty acid (PUFA) monoglycerides (MAGs) lies in the amphiphilic nature, and the beneficial health effects as PUFA precursors in various disorders including cancer, pulmonary hypertension, and inflammatory diseases. For exploring the potential therapeutic applications of these new amphiphilic lipids, particularly as main lipid constituents in the development of nanocarriers for delivery of drugs and PUFAs, it is of paramount importance to gain insight into their self-assembly behavior on exposure to excess water. This work describes the structural characteristics of self-assemblies based on two newly synthesized MAGs, namely docosahexaenoic acid (MAG-DHA) and docosapentaenoic acid (MAG-DPA) monoglycerides on exposure to excess water. We found that both lipids tend to form dominant inverse hexagonal (H2) phase in excess water at 25 °C and a temperature-triggered structural transition to an inverse micellar solution (L2 phase) is detected similar to that recently reported (A. Yaghmur et al., Langmuir, 2017, 33, 14045-14057) for eicosapentaenoic acid monoglyceride (MAG-EPA). An experimental SAXS structural evaluation study on the temperature-dependent behavior of these new monoglycerides is provided, and the effects of the unsaturation degree and the fatty acyl chain length on the self-assembled structural features in excess water and on the H2-L2 phase transition temperature is discussed. In addition, hexosomes stabilized by using the triblock copolymer F127 and the food-grade emulsifier citrem were investigated to gain insights into the effects of stabilizer and temperature on the internal nanostructure. These nanoparticles are attractive for use in the development of nanocarriers for delivering drugs and/or nutritional compounds as the beneficial health effects of ω-3 PUFA monoglycerides can be combined with loaded therapeutic agents or nutraceuticals.