Optical measurements reveal the preferential orientation of nanostructures within polymer films, which results from the fabrication process including mechanical and thermal treatments. As the wavelength of the incident light is generally much larger than the characteristic dimensions of the molecular arrangement in semi-crystalline or amorphous polymers, the optical signal originates not directly from the nanostructure of the polymers. Linear dichroism measurements were correlated with synchrotron radiation-based x-ray scattering data on commercially available polyetheretherketone (PEEK) thin films (12 to 50 μm). Annealing changed the structure of amorphous films to semi-crystalline ones associated with the measured linear dichroism. The intensity of the measured anisotropic signal depended on the film thickness. While for wavelengths between 450 and 1100 nm the transmission was higher when the polarizer was parallel to the machine direction, for larger wavelengths maximum transmission was observed with the polarizer perpendicular to the machine direction indicating excitations parallel and perpendicular to the PEEK molecule axis, respectively. Annealing PEEK films at temperatures between 160 and 240°C decreased the transmission at 540 nm by a factor of two, whereas the anisotropy remained constant. x-ray scattering revealed strongest anisotropy for a periodicity of 15 nm in the machine direction of the cast film extrusion process. The long-range order of amorphous and semi-crystalline entities can explain the x-ray scattering data and the related optical anisotropy of casted PEEK films.