Astragalus membranaceus is one of the important medicinal plant in China and Korea. It is used to increase metabolism and digestion, enhance the immune system, and promote the healing of wounds and injuries. In the present study, we used quantitative real-time PCR to investigate the expression of genes related to the biosynthesis of flavonoids, in addition to high-performance liquid chromatography to assess calycosin and calycosin-7-O-beta-D-glucoside accumulation, in the different plant organs of A. membranaceus. The transcript levels of all genes (AmPAL, AmC4H, Am4CL, AmCHS, AmCHR, AmCHI, AmIFS, AmI3'H, and AmUCGT) involved in calycosin and calycosin-7-O-beta-D-glucoside biosynthesis were the highest in the flower. Calycosin content was ordered as follows: leaf (145.56 mu g/g dry weight [DW]) > stem (18.3 mu g/g DW) > root (1.64 mu g/g DW) > flower (0.09 mu g/g DW), whereas calycosin-7-O-beta-D-glucoside content was ordered as follows: root (4.88 mu g/g DW) > stem (3.86 mu g/g DW) > leaf (2.0 mu g/g DW) > flower (not detected). All genes exhibited the highest transcription levels in the flower, whereas calycosin and its glycoside content were the highest in the leaf and root, respectively. Our results indicate that the enhancement of calycosin-7-O-beta-D-glucoside in the roots may originate from high calycosin accumulation in the stem and leaf. Thus, the mechanisms regulating calycosin and calycosin-7-O-beta-D-glucoside content differ in the different organs of A. membranaceus. The results are expected to provide baseline information from which the mechanism of flavonoid biosynthesis in the different organs of A. membranaceus may be elucidated.