Glycolipids form materials of considerable potential for a wide range of surfactant and thin film applications. Understanding the effect of glycolipid covalent structure on the properties of their thermotropic and lyotropic assemblies is a key step toward rational design of new glycolipid-based materials. Here, we perform molecular dynamics simulations of anhydrous bilayers of dodecyl β-maltoside, dodecyl β-cellobioside, dodecyl β-isomaltoside, and a C(12)C(10) branched β-maltoside. Specifically, we examine the consequences of chain branching and headgroup identity on the structure and dynamics of the lamellar assemblies. Chain branching of the glycolipid leads to measurable differences in the dimensions and interactions of the lamellar assembly, as well as a more fluid-like hydrophobic chain region. Substitution of the maltosyl headgroup of βMal-C(12) by an isomaltosyl moiety leads to a significant decrease in bilayer spacing as well as a markedly altered pattern of inter-headgroup hydrogen bonding. The distinctive simulated structures of the two regioisomers provide insight into the difference of ~90 °C in their observed clearing temperatures. For all four simulated glycolipid systems, with the exception of the sn-2 chain of the branched maltoside, the alkyl chains are ordered and exhibit a distinct tilt, consistent with recent crystallographic analysis of a branched chain Guerbet glycoside. These insights into structure-property relationships from simulation provide an important molecular basis for future design of synthetic glycolipid materials.