Oat ( L.) is an annual grass that has a high nutritional value and therapeutic benefits. β-glucan is one of the most important nutrients in oats. In this study, we investigated two oat varieties with significant differences in β-glucan content (high β-glucan oat varieties BY and low β-glucan content oat variety DY) during different filling stages. We also studied the transcriptome sequencing of seeds at different filling stages. β-glucan accumulation was highest at days 6-16 in the filling stage. Differentially expressed genes (DEGs) were selected from the dataset of transcriptome sequencing. Among them, three metabolic pathways were closely related to the biosynthesis of β-glucan by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, including xyloglucan:xyloglucosyl transferase activity, starch and sucrose metabolism, and photosynthesis. By analyzing the expression patterns of DEGs, we identified one gene and 32 transcription factors. Five modules were thought to be positively correlated with β-glucan accumulation by weighted gene co-expression network analysis (WGCNA). Moreover, the expression levels of candidate genes obtained from the transcriptome sequencing were further validated by quantitative real-time PCR (RT-qPCR) analysis. Our study provides a novel way to identify the regulatory mechanism of β-glucan synthesis and accumulation in oat seeds and offers a possible pathway for the genetic engineering of oat breeding for higher-quality seeds.