Major changes in tillage practices have occurred over the past 2 decades across the diverse range of soil types and rainfall zones that characterise cropping systems in southern Australia. However, there has been little corresponding change in the management of nutrients, especially phosphorus (P). This study investigated the effects of tillage and crop rotations on the stratification and transformation of P in soil profiles from 3 tillage/rotation trials encompassing 3 agro-ecological zones of southern Australia. Soil samples were collected from field trials at Longerenong (Vertosol, average rainfall 420 mm), Walpeup (Calcarosol, rainfall 325 mm), and Rutherglen (Chromosol, rainfall 650 mm) in Victoria. Soil samples from various depths were sequentially analysed for organic and inorganic P fractions. Phosphorus accumulated in the surface soil (0–0.1 m) across all sites and tillage practices/rotations studied but the proportion of P in different chemical fractions varied markedly among soil types and tillage practice/rotation. In the sandy Calcarosol, a greater proportion of fertiliser P was transformed into labile (resin-P) forms, whereas it tended to accumulate in non-labile pools in the finer textured Vertosol and Chromosol. The effects of tillage and crop rotation were generally confined to the topsoil with P strongly stratified in the topsoil in direct-drill and zero-tillage treatments compared with conventional tillage. The implications for management of P fertilisers in Victorian cropping systems are discussed.