Transmissible spongiform encephalopathies, induced by the accumulation of the pathological prion protein (PrPSc), impact various species including scrapie in small ruminants and Bovine Spongiform Encephalopathy in cattle. The characterization of isolates has transitioned from conventional to transgenic mice, but standardization is not yet available for all models and strains. This study, therefore, aims to evaluate the strain typing efficacy of two transgenic mouse models (TgshpIX and TgshpXI), overexpressing ovine PrP (ARQ/ARQ), and compare them with results from the RIII wild-type model. We examined the incubation period (IP), lesion, and PrPSc profiles resulting from intracerebral inoculation of well-defined sheep scrapie isolates (three classical: Langlade, Dawson and S805, and one atypical scrapie), classical BSE, and ovine BSE. Most isolates were successfully transmitted with an attack rate close to 100%, while in RIII, Langlade was barely and atypical scrapie was not transmissible. However, PrP genotype-dependent differences were evident with classical scrapie isolates. Notably, BSE triggered considerably longer incubation period in TgshpXI compared to TgshpIX. The incubation periods in RIII were significantly prolonged for all isolates. Lesion profiles were highly variable between the isolates and clearly distinguished only S805 in RIII. PrPSc profiles revealed distinctive patterns in both transgenic models, facilitating clear differentiation between each isolate and, most importantly, distinguishing both BSE isolates from scrapie. These findings are the basis for utilizing TgshpIX and TgshpXI mice in characterizing TSE isolates. Moreover, the results deepen our understanding of TSE strain diversity, employing ovine PrP-overexpressing transgenic mice as a valuable approach for biological prion strain typing.