Understanding the response of polyelectrolytes to their environment has challenged materials scientists for decades. Polyelectrolyte brushes are of particular interest, as they can regulate transport of ions and molecules, control wettability and adhesion of surfaces, or convert chemical and biochemical stimuli into optical, electrical, and mechanical signals. We combined neutron-reflectivity (NR), atomic force microscopy (AFM), surface forces apparatus (SFA) measurements, and coarse-grained molecular dynamics (MD) simulations to study the structure of planar biomimetic polyelectrolyte brushes (poly(styrenesulfonate), PSS) in a variety of solvent conditions, especially in the presence of multivalent counterions. Multivalent-ion induced lateral structural inhomogeneities of the PSS brushes were first indicated by NR measurements. AFM images provide a direct visualization of lateral inhomogeneities on the surface of polyelectrolyte brushes collapsed in solutions containing trivalent counterions. These images are interpreted in the context of a coarse-grained molecular model, and are corroborated by accompanying interaction-force measurements with the SFA. Our findings indicate that lateral inhomogeneities are absent from PSS brush layers collapsed in a poor solvent without multivalent ions The inhomogeneous structures of the polyelectrolyte brushes in the presence of multivalent ions can dramatically alter their functionalities in various biomedical applications, such as lubrication, sensors, and DNA-based microarrays.