Strain diversity in transmissible spongiform encephalopathies (TSEs) has been suggested to be “enciphered” in the structure of the misfolded prion protein isoform PrP^Sc. We have recently demonstrated the strain typing potential of the FT-IR spectroscopy technique, analyzing four different TSE agents adapted to Syrian hamsters [A. Thomzig, S. Spassov, M. Friedrich, D. Naumann and M. Beekes, Discriminating scrapie and BSE isolates by infrared spectroscopy of pathological prion protein J. Biol. Chem. 279 (2004) 33847-33854.] [1]. In the present paper, we have extended the FT-IR study, exploring the secondary structure, temperature stability, and hydrogen–deuterium exchange characteristics of PrP27–30, from the TSE agents 263K, ME7-H, 22A-H, and BSE-H. The strain differentiation capacity of the FT-IR approach was objectively proven for the first time by multivariate cluster analysis. The second derivative FT-IR spectra obtained from dried protein films or samples hydrated in H₂O or D₂O consistently exhibited strain-specific infrared characteristics in the secondary structure sensitive amide I region, complemented by strain dependent spectral traits in the amide II and amide A absorption regions, and the different H/D-exchange behaviour of the various PrP27-30 samples. FT-IR spectra of PrP27–30 samples from 263K, ME7-H and 22A-H exposed to increasing temperature (up to 90 °C) showed that a strain-specific response to heat treatment is associated with strain specific thermostability of distinct secondary structure elements, providing additional means for TSEs strain discrimination.