[Objective] To determine the typical RGS in Colletotrichum graminicola, and its signal peptide, transmembrane region and the secondary structure, and to provide a strong theoretical foundation for studying the positioning and function of RGS, but also provide an important theoretical guidance to clarify the other pathogen in Colletotrichum spp.. [Methods] Based on the four typical RGS sequences have been reported in Saccharomyces cerevisiae, to search RGS related protein sequence from the protein databases of Colletotrichum spp. with the BLASTp as well as the use of words, and to search the conserved domain in the SMART online. Meanwhile, bioinformatics analysis have made including the signal peptide, trans-membrane domain structure and the secondary structure, In addition, analysis of genetic relationships through comparative typical RGS in C. graminicola with homologous sequences of other species. [Results] There are six typical RGSs in C. graminicola, which have higher proportion of helical secondary structure. And these RGS do not contain the typical signal peptide sequence except for CgRGS6, and the half of six RGSs positioned in the nucleus, and the other is positioned in the plasma membrane, endoplasmic reticulum, and mitochondria, respectively. [Conclusion] C. higginsianum, C. gloeosporioides Cg-14/Nara gc5, C. orbiculare has a high sequence homology and close genetic relationship with C. graminicola.