Little is known about the structural basis of prion strains. Here we provide a high (3.0 Å) resolution cryo-electron microscopy-based structure of brain-derived fibrils of the mouse anchorless RML scrapie strain which, like the recently determined hamster 263K strain, has a parallel in-register β-sheet-based core. However, detailed comparisons reveal that variations in shared structural motifs provide a basis for prion strain determination.One-sentence summaryCryo-electron microscopy reveals a near-atomic structure of an infectious, brain-derived murine prion fibril and strain differences.