The installation of large scale colloidal nanoparticle thin films is of great interest in sensor technology or data storage. Often, such devices are operated at elevated temperatures. In the present study, we investigate the effect of heat treatment on the structure of colloidal thin films of polystyrene (PS) nanoparticles in situ by using the combination of grazing incidence small-angle X-ray scattering (GISAXS) and optical ellipsometry. In addition, the samples are investigated with optical microscopy, atomic force microscopy (AFM), and field emission scanning electron microscopy (FESEM). To install large scale coatings on silicon wafers, spin-coating of colloidal pure PS nanoparticles and carboxylated PS nanoparticles is used. Our results indicate that thermal annealing in the vicinity of the glass transition temperature T-g of pure PS leads to a rapid loss in the ordering of the nanoparticles in spin-coated films. For carboxylated particles, this loss of order is shifted to a higher temperature, which can be useful for applications at elevated temperatures. Our model assumes a softening of the boundaries between the individual colloidal spheres, leading to strong changes in the nanostructure morphology. While the nanostructure changes drastically, the macroscopic morphology remains unaffected by annealing near T-g.