Chitosan, trimethyl chitosan or polyethylenglycol-graft-trimethyl chitosan/DNA complexes were characterized concerning physicochemical properties such as hydrodynamic diameter, condensation efficiency and DNA release. Furthermore, cytotoxicity of polymers and uptake- and transfection efficiency of polyplexes were evaluated in vitro. Under conditions found in cell culture, formation of aggregates of approximately 1000 nm and strongly decreased DNA condensation efficiency was observed in the case of chitosan polyplexes. These characteristics resulted in only 7% cellular uptake in NIH/3T3 cells and low transfection efficiencies in 4 different cell lines. By contrast, quaternization of chitosan strongly reduced aggregation tendency and pH dependency of DNA complexation. Accordingly, cellular uptake was increased 8.5-fold compared to chitosan polyplexes resulting in up to 678-fold increased transfection efficiency in NIH/3T3 cells. Apart from reduction of the cytotoxicity, PEGylation led to improved colloidal stability of polyplexes and significantly increased cellular uptake compared to unmodified trimethyl chitosan. These improvements resulted in a significant, up to 10-fold increase of transfection efficiency in NIH/3T3, L929 and MeWo cells compared to trimethyl chitosan. This study not only highlights the importance of investigating polyplex stability under different pH- and ionic strength conditions but also elucidates correlations between physicochemical characteristics and biological efficacy of the studied polyplexes.