Despite the large number of possible glycerol-based phospholipids, biological membranes contain only a small number of them. For example, double bonds in acyl chains are preferably located in the sn-2 chain. The question that emerges is: Why? We have addressed this question through atomistic simulations by considering pure one-component bilayers comprising monounsaturated glycerophospholipids [1-stearoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (SOPC) and 1-oleoyl-2-stearoyl-sn-glycero-3-phosphatidylcholine (OSPC)] and membranes of these lipids mixed with cholesterol. By considering the cases in which an individual double bond is in either the sn-1 or the sn-2 chain, we elucidated how membrane properties depend on this intrinsic feature. We found small but systematic differences in all structural and dynamic membrane properties that we considered. It turns out that the differences are driven by two factors: the mismatch in the acyl chain lengths and the interaction of the double bond in the acyl chains with the cholesterol off-plane methyl groups. The results highlight the fact that unsaturated sn-2 chains lead to more disordered membranes than systems with unsaturated sn-1 chains. The differences between the two isomers are enhanced when cholesterol is present as a result of the interaction of the off-plane cholesterol methyl groups with the double-bond carbon segments in the lipid acyl chains.