Development of transgenic mouse models expressing heterologous prion protein (PrP) has facilitated and advanced in vivo studies of prion diseases affecting humans and animals. Here, novel transgenic mouse lines expressing a chimaeric murine/ovine (Mu/Ov) PrP transgene, including amino acid residues alanine, histidine and glutamine at ovine polymorphic codons 136, 154 and 171 (A136H154Q171), were generated to provide a means of assessing the susceptibility of the ovine AHQ allele to ruminant prion diseases in an in vivo model. Transmission studies showed that the highest level of transgene overexpression, in Tg(Mu/OvPrP(AHQ))EM16 (EM16) mice, conferred high susceptibility to ruminant prions. Highly efficient primary transmission of atypical scrapie from sheep was shown, irrespective of donor sheep PrP genotype, with mean incubation periods (IPs) of 154–178 days post-inoculation (p.i.), 100% disease penetrance and early Western blot detection of protease-resistant fragments (PrP(res)) of the disease-associated isoform, PrP(Sc), in EM16 brain from 110 days p.i. onwards. EM16 mice were also highly susceptible to classical scrapie and bovine spongiform encephalopathy (BSE), with mean IPs 320 and 246 days faster, respectively, than WT mice. Primary passage of atypical scrapie, classical scrapie and BSE showed that the PrP(res) profiles associated with disease in the natural host were faithfully maintained in EM16 mice, and were distinguishable based on molecular masses, antibody reactivities and glycoform percentages. Immunohistochemistry was used to confirm PrP(Sc) deposition in brain sections from terminal phase transmissible spongiform encephalopathy-challenged EM16 mice. The findings indicate that EM16 mice represent a suitable bioassay model for detection of atypical scrapie infectivity and offer the prospect of differentiation of ruminant prions.