Distinguishing the epimers iduronic acid (IdoA) and glucuronic acid (GlcA) has been a long-standing challenge for the mass spectrometry analysis of glycosaminoglycan (GAG) oligosaccharides. In this work, electron detachment dissociation (EDD) and Fourier transform ion cyclotron resonance mass spectrometry is shown to provide mass spectral features that can distinguish GlcA from IdoA in heparan sulfate (HS) tetrasaccharides. EDD of HS tetrasaccharide dianions produces a radical species that fragments to produce information-rich glycosidic and cross-ring product ions which can be used to determine the sites of acetylation/sulfation. More significantly, EDD of HS tetrasaccharide epimers produces diagnostic product ions that can be used to distinguish IdoA from GlcA. These diagnostic product ions are not observed in the tandem mass spectra obtained by collisionally activated dissociation or infrared multiphoton dissociation of the tetrasaccharides, suggesting a radical-initiated mechanism for their formation. Differences in the observed product ions obtained by EDD of the tetrasaccharide epimers can be rationalized by simple alpha-cleavage of an oxy radical located at C2 or C3 or a radical at C3 or C4. These radicals are proposed to arise from a hydrogen rearrangement in which a hydrogen atom is transferred from the C2 or C3 hydroxyl group or C3 or C4 to a carboxy radical at C5. This hydrogen transfer depends on the proximity of the carboxy radical to the hydroxyl group on C2 or C3 or the hydrogen on C3 or C4 and is thus influenced by C5 stereochemistry. These epimer-sensitive fragmentations should allow this approach to be applied to the structural analysis of a wide variety of GAG oligosaccharides.