Intracellular aggregation of tau protein in the brains of Alzheimer’s disease and related disorder cases heralds the onset of neurodegeneration and cognitive decline. Despite commonalities in cross-β-sheet structure, tau protomers adopt different conformations in the filament cores of each tauopathic disease. Moreover, brain-derived filaments differentially co-purify with noncovalent substances having probable anionic character. The ability of linear polyanions such as heparin to induce recombinant tau aggregation in vitro suggests that these interactions may induce filament formation while influencing their conformation depending on their structure and sites of interaction. Here we use Electron Paramagnetic Resonance (EPR) spectroscopy to probe tau aggregate conformation induced by a second class of polyanion represented by octadecyl sulfate (ODS) detergent. After incorporation of nitroxide-based spin labels at positions 298 and 311 of full-length human 2N4R tau, continuous wave (CW) and double electron-electron resonance (DEER) EPR modes were used to investigate their mobility and separation distances, respectively. Results indicate that ODS differentially affects tau aggregate conformation relative to heparin inducer, providing evidence that the nature of anionic inducer is a potential source of tau filament polymorphism in disease.