Nucleic acid drugs have great potential to treat many devastating aliments, but their application has been hindered by the lack of efficacious and nontoxic delivery vehicles. Here, a new library of poly(glycoamidoamine)s (D1-D4, G1-G4, and M1-M4) has been synthesized by polycondensation of esterified D-glucaric acid (D), dimethyl-meso-galactarate (G), and D-mannaro-1,4:6,3-dilactone (M) with diethylenetriamine (1), triethylenetetramine (2), tetraethylenepentamine (3), and pentaethylenehexamine (4). The stereochemistry of the carbohydrate hydroxyl groups and the number of amine units have been systematically changed in an effort to examine how the polymer chemistry affects the plasmid DNA (pDNA) binding affinity, the compaction of pDNA into nanoparticles (polyplexes), the material cytotoxicity, and the efficacy of nucleic acid delivery. The polymers with four secondary amines (D4, G4, and M4) between the carbohydrates were found to have the highest pDNA binding affinity and the galactarate polymers generally yielded the smallest polyplexes. Delivery studies with pDNA containing the firefly luciferase or β-galactosidase reporter genes in BHK-21, HeLa, and HepG2 cells demonstrated that all of the poly(glycoamidoamine)s deliver pDNA without cytotoxicity. Polymers D4, G4, and M4 displayed the highest delivery efficiency, where G4 was found to be a particularly effective delivery vehicle. Heparin competition assays indicated that this may be a result of the higher pDNA binding affinity displayed by G4 as compared to D4 and M4. Polyplexes formed by polymers with weaker pDNA affinities may dissociate at the cell surface due to interactions with negatively charged glycosaminoglycans, which would cause a decrease in the number of polyplexes that are endocytosed.