Sporadic Creutzfeldt-Jakob disease (sCJD) cases are currently subclassified according to the methionine/valine polymorphism at codon 129 of the PRNP gene and the proteinase K (PK) digested abnormal prion protein (PrP(res)) identified on Western blotting (type 1 or type 2). These biochemically distinct PrP(res) types have been considered to represent potential distinct prion strains. However, since cases of CJD show co-occurrence of type 1 and type 2 PrP(res) in the brain, the basis of this classification system and its relationship to agent strain are under discussion. Different brain areas from 41 sCJD and 12 iatrogenic CJD (iCJD) cases were investigated, using Western blotting for PrP(res) and two other biochemical assays reflecting the behaviour of the disease-associated form of the prion protein (PrP(Sc)) under variable PK digestion conditions. In 30% of cases, both type 1 and type 2 PrP(res) were identified. Despite this, the other two biochemical assays found that PrP(Sc) from an individual patient demonstrated uniform biochemical properties. Moreover, in sCJD, four distinct biochemical PrP(Sc) subgroups were identified that correlated with the current sCJD clinico-pathological classification. In iCJD, four similar biochemical clusters were observed, but these did not correlate to any particular PRNP 129 polymorphism or western blot PrP(res) pattern. The identification of four different PrP(Sc) biochemical subgroups in sCJD and iCJD, irrespective of the PRNP polymorphism at codon 129 and the PrP(res) isoform provides an alternative biochemical definition of PrP(Sc) diversity and new insight in the perception of Human TSE agents variability.