Abstract: To explore the regulatory mechanism of development and movement of grass stomata, on the basis of the primary clustering, the study successfully obtained independent and stable clusters of guard cells and subsidiary cells from single-cell transcriptome data in rice leaves by sub-clustering analysis of all the obtained epidermal cells when the resolution value of sub-clustering was set at 2.6, combined with more marker genes. They consist of 12 guard cells and 17 subsidiary cells, respectively. GO enrichment analysis revealed the biological processes of late stomatal development such as "microtubule-based movement", "signal transduction" and "response to stimulus". Gene regulatory network analysis revealed genes such as OsDOF11, OsMAPK2, OsNAC75, OsABCG4, OsERF52 and OsSWEET2b and their potential interaction networks. The results of this study provide a methodological reference for the identification of rare cell types in plants, and also provides new marker genes for the study of stomatal cells in other grasses.