We present an improved FISH strategy for differentiating the sex chromosomes of the dioecious model plant, Silene latifolia. Fixed mitotic protoplasts were dropped on a polyethylene naphthalate membrane, the X or Y chromosomes were isolated using nitrogen laser beam microdissection, catapulted by laser pressure, and amplified by DOP-PCR. A modified FAST-FISH protocol based on a short hybridization time combined with a low concentration of probe was used. The success of this approach is demonstrated by the differential labeling of the X and Y chromosomes and it could represent a quick method for comparing organization of plant genomes.