Abstract: In order to explore the genes related to crude protein synthesis in sorghum-sudangrass hybrid（Sorghum bicolor L.× S.sudanense P.）, the high crude protein（GH） and low crude protein（GL） sorghum-sudan-grass hybrid were used as materials in this study. The samples were collected at the peak jointing stage, and transcriptome sequencing（RNA-seq） was used for sequencing analysis. The results showed that a total of 1 491 di-fferentially expressed genes were identified from the two materials, compared with low crude protein（GL） mate-rials, the high crude protein（GH）material has 889（59.62%） up-regulated genes and 602（40.38%） down-regulated genes. Through gene ontology（GO） functional enrichment, it was found that the differentially expressed genes were significantly enriched in biological process（BP）, cell component（CC） and molecular function（MF）. The kyoto encyclopedia of genes and genomes（KEGG） enrichment analysis showed that the differentially expressed genes were mainly enriched in amino acid metabolism such as alanine and aspartic acid, nitrogen metabolism, porphyrin and chlorpohyll metabolism. A total of 25 differentially expressed genes related to sorghum-sudangrass hybrid crude protein synthesis were screened（20 genes up-regulated and 5 genes down-regulated）. Then, five differentially expressed genes were randomly screened for real-time quantitative PCR（RT-qPCR） verification, and the expression trend of the five genes was consistent with RNA-seq detection results. The results provided a reference for the study of the biosynthesis mechanism and secondary metabolic pathway of crude protein in sorghum-sudangrass hybrid, and lay a foundation for the further work of functional gene cloning and molecular marker development.