Ordered monolayers of polystyrene (PS) microspheres were prepared on both hydrophilic and hydrophobic substrates by convective self-assembly. Changes in the degree of ordering in the microsphere monolayers during annealing at temperatures below and above the glass transition temperature T-g of PS was quantitatively assessed using a customized image analysis method. This method overcomes the shortcomings of traditional Fourier transform methods for the analysis of images containing a small number of objects. In general, large ordered domains in the monolayers break up into smaller regions separated by cracks, due to thermal expansion of the polymer and the attractive dispersion force between microspheres. The substrate influences this phenomenon: the evolution was much slower for instance for monolayers on glass than for monolayers on PMMA substrates. In annealing experiments above Tg of PS, measurements of the widths of cracks in the monolayers showed differences between substrates: At later stages the width of the cracks in monolayers on PS substrates were observed to decrease more quickly than for monolayers on PMMA substrates. After very long times at relatively high temperatures, the monolayers on both substrates became essentially smooth films.