In order to understand the genetic law and formation mechanism of black and yellow prosomillet seeds, this study used yellow seed coat variety "Huang limi I" and black seed coat variety "2016106I" to construct F₂ generation population, analyze the genetic law of seed coat color, use F₃ generation seeds for transcriptome sequencing, and explore the key genes that affect the color of millet seeds. The results showed that: (1) Through the hybrid combination of yellow seed millet I × Genetic analysis of seed coat color of F₁ and F₂ populations of 2016106I hybrid combination showed that black was dominant to yellow and was controlled by a single gene. (2) According to the results of GO and KEGG enrichment analysis, a total of 13 differentially expressed genes related to flavonoid synthesis pathway were screened, only C2845_ PM02G08740、C2845_ PM04G29280 and C2845_ PM09G22680 is an up-regulated gene, and the other 10 are down-regulated genes. Among them, C2845 is the gene with the largest differential multiple_ PM04G29280, the multiple of difference is 4.88 times; These genes encode many enzymes such as cinnamyl alcohol dehydrogenase and cinnamyl CoA reductase, which are closely related to the formation of seed coat color. However, the major genes related to the color formation of the seed coat of millet can be determined only after further study. This study provides a theoretical basis for clarifying the mechanism of color formation of seed coat and breeding of new varieties of prosomillet.