[Objective] Using Agrobacterium-mediated transformation as a tool, we built a stable genetic transformation of Hypsizygus marmoreus. [Methods] After binary expression vector pYN6982 was transformed into Agrobacterium tumefaciens LBA4404, we transformed H. marmoreus SIEF3133 blended vegetative dikaryotic mycelia via Agrobacterium-mediated transformation. [Results] Dozens of transgenic strains of H. marmoreus with genetic stability were obtained after hygromycin resistance selection, PCR identification and mitotic stability test. By fluorescence microscopy analysis of randomly selected transformants, green fluorescence can be observed. This enhanced green fluorescent protein gene (egfp) has expressed in transgenic H. marmoreus. Moreover, two of eight randomly selected H. marmoreus transformants contain kanamycin gene (kan) sequences from outside the transferred DNA (T-DNA) border repeats using PCR analysis. [Conclusion] Transgenic strains of H. marmoreus with genetic stability and egfp expression were obtained. Also, DNA sequences from beyond the classically defined T-DNA border direct repeats can be transferred into the H. marmoreus mycelia. This phenomenon requires attention in H. marmoreus functional gene study.