A method for visualization of fungal nuclear structures using the DNA specific fluorochrome SYTOX Green and confocal laser scanning microscopy is presented. The staining procedure detects nuclei in different cell types with high contrast. Furthermore, the method permits generation of high-resolution confocal image stacks using optimised scanning conditions. This approach significantly improved visualization of stained fungal nuclear structures. The technique was applied to some lichenized fungi, but can also be used to study similar detail in non-lichenized fungi. In certain cases, incompletely separated chromosome-like structures, heterochromatic material as well as weakly stained nucleolar regions can be seen. In Xanthoria parietina, variation in the nuclear division was observed in developing ascospores. Delayed division after the formation of endospore thickening resulted in the exchange of nuclear material through a narrow isthmus.