Mammalian prion diseases involve conversion of normal prion protein, PrP^C, to a pathological aggregated state (PrP^res). The three-dimensional structure of PrP^res is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent β-sheet content. PrP^res molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP^res with GPI- and glycan-deficient PrP^res produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP^res revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to β-sheets. These PrP^res spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP^res were nearly indistinguishable. With ME7 PrP^res, modest differences between the wild-type and anchorless spectra were detected, notably an ∼2 cm^–1 shift in an apparent β-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP^res, at least relative to the differences observed between strains, but can subtly affect turns and certain β-sheet components. Recently reported H–D exchange analyses of anchorless PrP^res preparations strongly suggested the presence of strain-dependent, solvent-inaccessible β-core structures throughout most of the C-terminal half of PrP^res molecules, with no remaining α-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP^res.