A new di-O-prenylated isoflavone, 5,7-di-O-prenylbiochanin A (1), together with three known compounds, 7-O-methylglabranin (2), tephrowatsin C (3) and flemichapparin B (4), were isolated from the stems of Tephrosia tinctoria. The structures of these compounds were elucidated by extensive 2D NMR spectral studies.