Plants belonging to the genus Miscanthus are considered promising bioenergy crops. Here, we report the establishment of tissue culture system through particle bombardment-mediated transformation in Miscanthus sinensis Anderss. Callus was induced efficiently from mature seeds in a medium containing a combination of a relatively high level of 2,4-dichlorophenoxyacetic acid (5 mg L−1) and a relatively low level of 6-benzyladenine (BA) (0.01 mg L−1). Callus induction potential of 18 accessions of M. sinensis, which were collected from various sites in Japan, was compared. Significant correlation was detected between compact (embryogenic) callus induction frequency and average annual air temperature in collection sites. An accession from Tanegashima Island showed the highest production of compact callus. We found that a higher level of BA causes callus browning; the 2 mg L−1 BA is the optimal concentration for regeneration. Both compact and friable calli were suitable for particle bombardment transformation. Through selection under the presence of 50 mg L−1 hygromycin for 3 weeks and further selection under the presence of 150 mg L−1 for 1 month, hygromycin-resistant calli survived, of which 72.2% had been entirely transformed. Plants were regenerated from calli in the presence of hygromycin; transcripts of the hpt and gfp genes, which were cobombarded to the calli, were detected in the regenerated plants. This is the first report on the establishment of the in vitro culture of M. sinensis using mature seeds, the variation of callus formation among accessions collected from various sites in Japan, and particle bombardment-mediated transformation in the genus Miscanthus.