Plastid transformation has been successfully employed in several dicotyledonous plants for genetic modification, but it is difficult to be achieved in monocotyledonous cereal crops and remains still a demanding technique in the engineering agriculture field. One of major obstacles applying plastid transformation in crop plants is the limited availability of suitable selective markers. Hygromycin B is an aminoglycoside antibiotic which has the ability to inhibit prokaryotic protein synthesis, but to our best knowledge, it has never been reported in the research of plastid transformation yet. In this study, we aimed to use the hygromycin phosphotransferase gene (hpt) as a selective marker gene for tracking plastid transformation in rice. A site-specific transformation vector of rice plastid, pCTE04, was constructed for dicistronic expression of the hygromycin phosphotransferase (hpt) and enhanced green fluorescent protein genes (EGFP). The results showed that transplastomic rice plants successfully grew up after a gradual selection of hygromycin resistance. Integration of the dicistronic expression cassette into the intergenic region trnI-trnA of plastid genome was verified by Southern blotting analysis. Transplastomic expression of the hpt and EGFP genes was identified by RT-PCR. The stable expression of dicistronic cassette in the rice was confirmed by confocal microscopy. Although the plastid transformants in rice presented a heteroplastomic status, this study may facilitate the development of new selective markers and further improvement of plastid transformation efficiency in cereal crops.