The number of double bonds in the acyl chains of membrane lipids varies tremendously at all scales of life, from the organism level to the subcellular level, where differences in lipid unsaturation can be observed between two membrane leaflets or between two continuous regions of the same organelle. Here, we review different approaches that have been used to understand the variability in the acyl chain composition of lipid membranes. We suggest that a full understanding of lipid unsaturation is limited not only by technical difficulties but also because some properties afforded by unsaturated lipids in membrane lipids are likely to be subtler than a mere effect on 2D fluidity, notably, the way the position of double bonds in the acyl chains affect the motion of transmembrane proteins, the adsorption of peripheral proteins, or some mechanical properties of the membrane.