The coexistence of liquid ordered Lo and liquid disordered Ld phases in synthetic and plasma membrane-derived vesicles serves as a model for biomembrane heterogeneity. However, the connection between the structures of microscopic phases present in vesicles at low temperatures and the tiny ordered "raft" domains of biomembranes at body temperature is unclear. To study the Lo phase structure across temperatures, we performed atomistic molecular dynamics simulations, differential scanning

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... ry, and fluorescence spectroscopy on the Lo phase in binary and ternary lipid mixtures. Our results reveal an Lo phase with highly ordered and hexagonally packed clusters of saturated lipid chains at low temperatures. These clusters melt upon heating, and numerous membrane properties reflect this transition as two regimes with different temperature dependence. Still, the transition between the regimes is continuous, and they both match the description of the Lo phase with high order and relatively high mobility. Our findings question the use of vesicles displaying Lo–Ld coexistence as models for heterogeneity in cellular membranes, as they likely correspond to different molecular organizations.