Self-assembly is a key process in forming biological materials. Especially the interaction between amphiphiles and polyelectrolytes has been widely investigated in recent years due to their potential application in industry and medicine, with a special focus on gene therapy. Accordingly, we investigated the formation of lipoplexes by mixing the cationic lipid DOTAP (1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)) with different anionic polyelectrolytes (PE), such as NaPA (sodium polyacrylate), CMC (sodium carboxymethyl cellulose) with different degrees of substitution (DS, namely, different charge density), PSS (sodium polystyrenesulfonate) and DNA (deoxyribonucleic acid sodium salt). The goal of this project was to explore the influence of different system parameters, such as the charge ratio, CR = [+]/[-] = [DOTAP]/[PE], the charge density of the PE, or the type of PE on the morphology of the formed complexes. The investigation of these systems was performed by cryo-transmission electron microscopy (cryo-TEM), and with small-angle X-ray scattering (SAXS), to support our findings. In our experiments, we obtained a comprehensive picture of the formed lipoplexes, and how their structure depends on the different properties of the employed polyelectrolyte. Although the basic nanostructure of all complexes is lamellar, their detailed morphology depends strongly on parameters of the PE, e.g., the persistence length, charge density, or the polymer backbone. Understanding these specific interactions will allow the formation of more stable and optimized complexes as they are needed for drug or genetic material delivery.