The complex sphingolipids exhibit a diversity of ceramide acyl chain structures that influence their trafficking and intracellular distributions, but how the cell discerns among the different ceramides to affect such sorting remains unknown. To address mechanism, we synthesized a library of GM1 glycosphingolipids with naturally varied acyl chains and quantitatively assessed their sorting at the plasma membrane and among different endocytic pathways. We found that a stretch of at least 14 saturated carbons extending from C1 at the water-bilayer interface dictated nanodomain assembly and lysosomal sorting by exclusion from endosome sorting tubules. Sorting by the C14*-motif was cholesterol dependent. Perturbations of the C14*-motif by unsaturation or reduced acyl chain length enabled GM1 entry into endosomal sorting tubules of the recycling and retrograde pathways independently of cholesterol and led to exclusion from plasma membrane nanodomains. Unsaturation occurring beyond the C14*-motif in very long acyl chains however rescued nanodomain assembly and lysosomal sorting. These results define a structural motif underlying membrane organization of sphingolipids and implicate cholesterol-sphingolipid nanodomain formation in sorting mechanisms.