Assembly morphology made from lipids is controlled by the balance between the polar headgroup and the hydrophobic tails. In this study, we showed the various generations of polyamidoamine dendron-bearing lipids could form various assembly morphologies. Furthermore, the effect of the assembly morphologies made from dendron-bearing lipids for transfection abilities were examined. We synthesized three novel dendron-bearing lipids, DL-U2-G1 (G1), DL-U2-G2 (G2), and DL-U2-G3 (G3), which included first, second, and third generation polyamidoamine dendrons, respectively. Transmission electron microscopy showed that lipoplexes (complexes with cationic lipids and plasmid DNA) comprising G1 had multilamellar structures. G2 presented as aggregates of cubic particles and G3 exhibited clusters of spherical micelles. The ability to form complexes with plasmid DNA was in the decreasing order G3 > G2 > G1; calcein release from endosomes was in the order G3 > G2, G1; and transfection activity followed the order G1 > G2, G3. Interaction of the lipoplexes with heparin suggests that G3 had a lower level of plasmid DNA dissociation from lipoplexes than G1 in vitro. These results suggest that the size of the DL-U2 headgroup determines assembly morphology and that the structure markedly affects transfection activity.