Mammalian prions (PrP^Sc) consist of misfolded, conformationally altered, self‐replicating states of the sialoglycoprotein called prion protein or PrP^C. Recent studies revealed that the sialylation status of PrP^Sc plays a major role in evading innate immunity and infecting a host. Establishing the type of linkage by which sialic acid residues are attached to galactose is important, as it helps to identify the sialyltransferases responsible for sialylating PrP^C and outline strategies for manipulating the sialyation status of PrP^Sc. Using enzymatic treatment with sialidases and lectin blots, this study demonstrated that in N‐linked glycans of PrP^Sc, the sialic acid residues are predominantly alpha 2,6‐linked. High percentages of alpha 2,6‐linked sialic acids were observed in PrP^Sc of three prion strains 22L, RML, and ME7, as well as PrP^Sc from brain, spleen, or N2a cells cultured in vitro. Moreover, the variation in the percentage of alpha 2,3‐ versus 2,6‐linked sialic acid was found to be relatively minor between brain‐, spleen‐, or cell‐derived PrP^Sc, suggesting that the type of linkage is independent of tissue type. Based on the current results, we propose that sialyltransferases of St6Gal family, which is responsible for attaching sialic acids via alpha 2,6‐linkages to N‐linked glycans, controls sialylation of PrP^C and PrP^Sc.