Licorice is a frequently-used medicinal plant worldwide. Two triterpenoids, 18α-glycyrrhizic acid (18α-GC) and 18β-glycyrrhizic acid (18β-GC), are the key medicinal components accumulated in licorice. Biosynthesis of triterpenoids is a complex process that involves many secondary metabolic pathways. In this study, we tried to identify the key enzymes and pathways for the triterpenoid biosynthesis in licorice by analyzing the gene expression patterns in samples containing different GC levels. Glycyrrhizia glabra (one of the original species used as licorice in Chinese Pharmacopoeia) seeds were irradiated by X-ray and cultivated for one year, and samples with different GC contents were selected by HPLC analysis. RNA-Seq was performed to determine the gene expression in three X-ray irradiated G. glabra samples (H1, H2, and H3) with the highest GC content and one control G. glabra sample (L1) with the lowest GC content. 28.44 Gb raw data was generated and 45.4 million, 43.3 million, and 45.9 million clean reads were obtained in samples H1, H2, H3, and L1, respectively. Approximately 48.53 % of genes were annotated searching against GO and KEGG databases. A total of 1376 core differentially expressed genes (DEGs) were identified, which mainly enriched in phenylpropanoid metabolism, glycometabolism, plant circadian rhythm, and terpenoid biosynthetic pathway. 15 core DEGs selected from the 1376 DEGs were further verified by qRT-PCR, which confirmed that the RNA-Seq results were accurate and reliable. This study provides a basis for future functional genes mining and molecular regulatory mechanism elucidation of triterpenoid biosynthesis in licorice.