Seasonal variations in contents of bioactive markers in Australian-grown Salvia miltiorrhiza roots were investigated in a two-year field trial. Cryptotanshinone, tanshinone I, tanshinone IIA, and salvianolic acid B were quantitatively determined by reversed-phase (RP) HPLC. Similar accumulation patterns were observed for the three tanshinones throughout the trial period, although roots harvested in the first year was found to contain relatively higher contents of these compounds. In contrast, the content of salvianolic acid B was peaked at 250 days after planting in the first year, and subsequently maintained at a plateau level in the second-year period. Linear correlations between the contents of individual tanshinones were observed, but not between those of tanshinones and salvianolic acid B. The findings suggest that tanshinones and salvianolic acid B have different accumulation patterns in Australian-grown Salvia miltiorrhiza roots, which should be critically considered for optimum harvesting of the roots for pharmaceutical applications.