Transmissible spongiform encephalopathies (TSEs) are a group of zoonotic and fatal neurodegenerative disorders that affect humans and animals. The pathogenesis of TSEs involves a conformational conversion of the cellular prion protein (PrP) into abnormal isoforms. Currently, cellular and pathological forms of PrP are differentiated using specific antibody-based analyses that are resource intensive and not applicable to all species and strains. Thus, there is an urgent need for sensitive and efficient assays that can detect pathological forms of PrP. Using systematic evolution of ligands by exponential enrichment, we developed DNA aptamers that can differentiate normal and abnormal PrP isoforms. These aptamers represent the first reagents that can identify pathological isoforms of PrP across multiple host species. Second, they are able to distinguish different strains of prions. Third, they can be used to detect prions in peripheral blood cells, which are otherwise undetectable using conventional antibody-based detection methods. Thus, DNA aptamers offer promise for the development of presymptomatic screens of tissue, blood and other body fluids for prion contamination. © 2011 by the Society for Experimental Biology and Medicine.