The hydrophobic moiety of cationic amphiphiles plays an important role in the transfection process because its structure has an impact on both the type of the supramolecular assembly and the dynamic properties of these assemblies. The latter have to exhibit a compromise between stability and instability to efficiently compact then deliver DNA into target cells. In the present work, we report the synthesis of new cationic amphiphiles featuring a thioether function at different positions of two 18-atom length lipid chains and we study their physicochemical properties (anisotropy of fluorescence and compression isotherms) with analogues possessing either oleyl (C181) or stearyl (C180) chains. We show that the fluidity of cationic lipids featuring a thioether function located close to the middle of each lipid chain is intermediate between that of oleyl- and stearyl-containing analogues. These properties are also supported by the compression isotherm assays. When used as carriers to deliver a plasmid DNA, thioether-containing cationic amphiphiles demonstrate a good ability to transfect human-derived cell lines, with those incorporating such a moiety in the middle of the chain being the most efficient. This work supports the use of a thioether function as a possible alternative to unsaturation in aliphatic lipid chains of cationic amphiphiles to modulate physicochemical behaviours and in turn biological activities such as gene delivery ability.