The assembly of tau into amyloid filaments is associated with more than 20 neurodegenerative diseases, collectively termed tauopathies. Electron cryo-microscopy (cryo-EM) structures of brain-derived tau filaments revealed that specific structures define different diseases, triggering a quest for the development of experimental model systems that replicate the structures of disease. Here, we describe 12 phosphomimetic serine/threonine-to-aspartate mutations in tau, which we term PAD12, that collectively induce the in vitro assembly of full-length three-repeat tau into filaments with the same structure as paired helical filaments extracted from the brains of individuals with Alzheimer’s disease. Solution-state nuclear magnetic resonance spectroscopy suggests that phosphomimetic mutations in the carboxy-terminal domain of tau may facilitate filament formation by disrupting an intramolecular interaction between two IVYK motifs. PAD12 tau can be used for both nucleation-dependent and multip