To obtain a genetic marker to observe and study the interaction of Sclerotinia sclerotiorum with its hosts, isolates ND30 and ND21 were transformed using pCT74 and gGFP constructs, both containing genes for the green fluorescent protein (GFP) and hygromycin B phosphotransferase. Putative transformants were obtained using polyethylene glycol-mediated transformation of protoplasts. Seven stable gfp transformants were identified and evaluated for fluorescence in vitro and in planta, pathogenicity and colonization of host tissues. Real-time quantitative polymerase chain reaction detected a single copy of the gfp gene in transformants. Fluorescence was quantified directly from mycelium and protein extracted from hyphae. The seven transformants (four from ND30 and three from ND21) were pathogenic on dry bean, canola, soybean and sunflower. However, depending on the host, three transformants differed significantly (P = 0·05) in the length of lesions formed compared to the wild-type. Hyphae fluoresced in plant tissue and could clearly be distinguished from plant cells. Infection and colonization of tissues were clearly visible with a fluorescent microscope. Transformants differed in the intensity of GFP expression both in vitro and in planta.