Heparan sulfate (HS) is a linear polysaccharide covalently attached to proteoglycans on cell surfaces and within extracellular matrices in all animal tissues. Many biological processes are triggered by the interactions among HS binding proteins and short structural motifs in HS chains. The determination of HS oligosaccharide structures using liquid chromatography-mass spectrometry (LC-MS) is made challenging by the existence of positional sulfation and acetylation isomers. The determination of uronic acid epimer positions is even more challenging. While hydrophilic interaction liquid chromatography (HILIC) separates HS saccharides based on their composition, there is a very limited resolution of positional isomers. This lack of resolution places a burden on the tandem mass spectrometry step for assigning saccharide isomers. In this work, we explored the use of the ion mobility dimension to separate HS saccharide isomers based on molecular shape in the gas phase. We showed that the combination of HILIC and cyclic ion mobility mass spectrometry (cIM-MS) was extremely useful for resolving HS positional isomers including uronic acid epimers and sulfate positions. Furthermore, HILIC-cIM-MS differentiated multicomponent HS isomeric saccharide mixtures. In summary, HILIC-cIM-MS provided high-quality data for analysis of HS oligosaccharide isomeric mixtures that may prove useful in the discovery of new structural motifs for HS binding proteins and for the targeted quality control analysis of commercial HS products.