The effect of the molecular organization of lipid components on the properties of the bilayer membrane has been a topic of increasing interest. Several experimental and theoretical studies have suggested that cholesterol is not randomly distributed in the fluid-state lipid bilayer but assembles into nanoscale domains. Several cholesterol-enriched nanodomain structures have been proposed, including rafts, regular or maze arrays, complexes, and superlattices. At present, the molecular mechanisms by which lipid composition influences the assembly and stability of lipid nanodomains remain unclear. In this study, we have used molecular dynamics (MD) simulations to investigate the effects of the molecular organization of cholesterol-superlattice versus random-on the structure of and interactions between lipids and water in lipid bilayers of cholesterol and 1-palmitoyl-2-oleoyl-phosphatidylcholine (cholesterol/POPC) at a fixed cholesterol mole fraction of 0.40. Based on four independent replicates of 200-ns MD simulations for superlattice or random bilayer, statistically significant differences were observed in the lipid structural parameters, area per lipid, density profile and acyl chain order profile, as well as the hydrogen bonding between various pairs (POPC and water, cholesterol and water, and POPC and cholesterol). The time evolution of the radial distribution of the cholesterol hydroxy oxygen suggests that the lateral distribution of cholesterol in the superlattice bilayer is more stable than that in the random bilayer. Furthermore, differences in the lipid diffusivity between the two structures suggests overall bilayer organization is important in the assembly and stability of the superlattice nanodomains.