The development of safe and efficient gene delivery vectors is an essential prerequisite for successful gene therapy. As viral vectors suffer from inherent disadvantages, cationic polymers as non-viral vectors have great potential in gene delivery, but their practical application so far is seriously hampered due to their relatively low transfection efficiency caused by multiple extra- and intracellular gene delivery barriers. Therefore, it is important to provide cationic polymers with functionalities that can seriously influence polymeric properties which are important to overcome gene delivery barriers. In this paper, we aim to contribute to the understanding of the effect of functionalities in cationic polymers on their gene delivery properties and transfection activity. As poly(amido amine)s can be easily provided with a large variety of chemical functionalities, we have focused on this class of cationic polymers. It is shown that various structural characteristics in these peptidomimetic polymers such as charge density, rigidity, basicity, hydrophilicity/hydrophobicity, degradability and type of amino groups influence one or more gene delivery properties such as DNA binding capability, colloidal stability, endosomal escape (buffer capacity), vector unpacking, cytotoxicity, and eventual transfection efficiency. Optimal combination of the functionalities in the poly(amido amine)s may lead to significant increase of the level of gene expression. This indicates that multifunctionalized polymers like the poly(amido amine)s can evolve to the next generation of non-viral gene delivery system for gene therapy.