Agrobacterium-mediated transformation was conducted in order to generate DNA insertional mutants of Flammulina velutipes. Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into gill tissues of Flammulina velutipes strain KACC42777. The transformants resistant on hygromycine (30 µg/ml) were confirmed by PCR. The targeted insertional sites were amplified by inverse PCR and sequenced. To find the phenotype variation of all generated transformants, bottle cultivation which followed by the standard cultivation protocol were conducted. Color variation was observed on the cultivated fruiting bodies. Futhermore, the transformant pool will be used as a good genetic resources for studying gene function.