The outcrossing rate of sterile lines plays an important role in hybrid seed production. In this study, two soybean cytoplasmic male sterile lines with significant difference in outcrossing rate were studied, and their ma? ture flower buds at full bloom stage were tested. Transcriptome sequencing was performed. After comparative analy? sis, 1925 differential genes were screened out. Based on GO classification, 1361 differential genes were annotated into the 30 branches of the biological processes, cell components and molecular functions, main biological process? es, catalytic activities, oxidoreductase activities, redox process and carbohydrate metabolism process. 864 differen? tial genes were annotated into 114 metabolic pathways by KEGG classification, including metabolic pathway path? ways, biosynthetic pathways of secondary metabolites, and phytohormone signal transduction pathways, including 13 secondary metabolic pathways, main phenylpropanoid biosynthesis and flavonoid biosynthesis. According to sig? nificant value of gene difference and the number of enriched genes, the pathways of flavonoid biosynthesis were ana? lyzed, gene FLS was screened as the key to flower color. Previous report showed the flower color affected induce? ment insect to transfer pollen, which further influenced outcrossing rate. This study was to elucidate the biosynthetic pathways affecting soybean outcrossing rate and clarify the molecular mechanism of soybean outcrossing rate.