Single molecule spectroscopy is used to obtain detailed information on the photophysical properties of immobilized perylenediimide-based molecules, substituted in the bay positions. The fluorescence spectra recorded for numerous single molecules show a clear bimodal distribution of the peak position. Within the low energy component of the distribution, two different vibronic shapes of the emission spectrum can be seen, which can be correlated to different decay times, We show that former observation can be explained by conformational changes of the bay substituents while the latter are related to twisting of the single molecule around the central perylenediimide long axis.