Fluorescence resonance energy transfer (FRET) is a technique now widely applied to probe biological and other complex systems for the determination of fluorophore separation and structure. Recently the theory behind the anisotropy of fluorescence has been extended to include the case of a residual polarization following energy transfer between fluorophores, and here the theory is further extended to accommodate two-photon excitation. This reveals not only novel polarization characteristics but also a distance dependence whose analysis does not require a priori knowledge of the donor–acceptor spectral overlap. The two-photon FRET anisotropy results mirror their one-photon counterparts, in terms of fluorophore separation characteristics and also their relationship to the anisotropies for isolated fluorophores. Moreover, the two-photon results are not restricted to a plane polarized input, results being given for both plane and circularly polarized pump radiation.