The mid-lithospheric discontinuity (MLD) is a seemingly sharp decrease in seismic velocity at depths internal to the lithosphere and appears to be a pervasive feature beneath continental interiors. Its presence within cratons, which have remained relatively stable since formation, suggests that the MLD may result from processes associated with continent formation. We use P- to S-wave receiver functions to interrogate seismic anisotropy across the MLD within the ca. 1.35–1.55 Ga Granite-Rhyolite Province of the central United States. Our analysis reveals strong evidence for sharp changes in the orientation of anisotropy across multiple MLDs, with an approximately north to northwest fast orientation of anisotropy in the upper lithosphere. The consistency of this signature over a large region suggests that the observed anisotropy is a relic of North American craton formation. In addition, the presence of several distinct anisotropic layers within the cratonic lithosphere supports models for craton formation via stacked subducted slabs or a series of underthrusting events.