Abstract Five bituminous samples were carefully studied by confocal laser scanning microscopy using 488 nm excitation radiation and observing 500–530 nm of emission. The images revealed the microstructure of bitumen. The influence of the admixture of mineral aggregates concerning the microstructure was tested. For the minerals, no significant influence was found. For understanding the origin of fluorescent signals, the samples were separated into asphaltenes and maltenes and analyzed with fluorescence spectroscopy. Although former works have assumed the origin of fluorescent emissions in bitumen to be found in the asphaltene fraction, the asphaltenes produce little to no emissions, but the maltenes exhibit strong fluorescence in the observed spectral region. For deeper insight, fractionation of the bitumina into the SARA fractions by chromatographic column separation was necessary. The fluorescence spectra of these fractions were analyzed and revealed the aromatics and resin phases to be the only components capable of sufficiently intense fluorescent emission. This is a strong argument for a complex internal microstructure consisting of a mantle of aromatics surrounding an inner core.