Astragalus membranaceus var. mongholicus (AMM), a member of the Leguminosae, is one of the most important medicinal plants worldwide. The dried roots of AMM have a wide range of pharmacological effects and are a traditional Chinese medicine. Here, we report the first chromosome-level reference genome of AMM, comprising nine pseudochromosomes with a total size of 1.47 Gb and with the total number of 27,868 protein-encoding genes. Comparative genomic analysis reveals that AMM has not experienced an independent whole-genome duplication (WGD) event after the WGD event shared by the Papilionoideae species. Analysis of the long terminal repeat retrotransposons (LTR-RTs) suggests a recent burst of these elements at approximately 0.13 million years ago (Mya), which may explain the large size of the AMM genome. Moreover, multiple gene families involved in the biosynthesis of triterpenoid and flavonoid were expanded, and our data indicate that tandem duplication is the main driver for the expansion of these families. Among the expanded families, the phenylalanine ammonia-lyase (PAL) gene family was primarily expressed in the roots of AMM, suggesting their roles in the biosynthesis of phenylpropanoid compounds. Furthermore, the functional versatility of 2,3-oxidosqualene cyclase (OSC) genes in cluster Ⅲ may play a critical role in the diversification of triterpenoids in AMM. Our findings provide novel insights into understanding the triterpenoid and flavonoid biosynthesis and could facilitate research on the genetics and medical applications of AMM in the future.Copyright © 2022. Published by Elsevier Inc.