We investigate two-dimensional (2D) assembly of the icosahedral turnip yellow mosaic virus (TYMV) under cationic lipid monolayers at the aqueous solution–vapor interface. The 2D crystallization of TYMV has been achieved by enhancing electrostatically induced interfacial adsorption, an approach recently demonstrated for another virus. In situ X-ray scattering reveals two close-packed 2D crystalline phases of TYMV that are distinct from the previously reported hexagonal and centered square arrays of TYMV. One of the newly observed phases arises from either a dimeric double-square (2 × 1) or tetrameric square (2 × 2) unit cell. The other is a rhombic crystal with a lattice angle of 80°. The two observed crystal phases are substantially less dense (by over10%) than a 2D lattice of TYMV could be according to its known size and shape, indicating that local anisotropic interparticle interactions play a key role in stabilizing these crystals. TYMV's anisotropy attributes and numerical analysis of 2D arrays of virus-shaped particles are used to derive a model for the rhombic crystal in which the particle orientation is consistent with the electrostatic lipid–TYMV attraction and the interparticle contacts exhibit steric complementarity. The interplay between particle anisotropy and packing is contrasted between the rhombic crystal model and the square crystal. This study highlights how the high symmetry and subtle asphericity of icosahedral particles enrich the variety and complexity of ordered 2D structures that can be generated through self-assembly.