Poly(ethylene glycol) (PEG) hydrogels provide a versatile platform to develop cell instructive materials through incorporation of a variety of cell adhesive ligands and degradable chemistries. Synthesis of PEG gels can be accomplished via two mechanisms: chain and step growth polymerizations. The mechanism dramatically impacts hydrogel nanostructure, whereby chain polymerized hydrogels are highly heterogeneous and step growth networks exhibit more uniform structures. Underpinning these alterations in nanostructure of chain polymerized hydrogels are densely-packed hydrophobic poly(methyl methacrylate) or poly(acrylate) kinetic chains between hydrophilic PEG crosslinkers. As cell-material interactions, such as those mediated by integrins, occur at the nanoscale and affect cell behavior, it is important to understand how different modes of polymerization translate into nanoscale mechanical and hydrophobic heterogeneities of hydrogels. Therefore, chain- and step-growth polymerized PEG hydrogels with macroscopically similar macromers and compliance (e.g., methacrylate-functionalized PEG (PEGDM), MW=10 kDa and norbornene-functionalized 4-arm PEG (PEGnorb), MW=10 kDa) were used to examine potential nanoscale differences in hydrogel mechanics and hydrophobicity using atomic force microscopy (AFM). It was found that chain-growth polymerized network yielded greater heterogeneities in both stiffness and hydrophobicity as compared to step-growth polymerized networks. These nanoscale heterogeneities impact cell-material interactions, particularly human mesenchymal stem cell (hMSC) adhesion and spreading, which has implications in use of these hydrogels for tissue engineering applications.