A new sequential extraction scheme for acid sulfate soil materials has been evaluated for iron partitioning in a range of synthetic iron-bearing minerals and natural acid sulfate soil materials. This sequential extraction procedure employs six steps to quantify (1) exchangeable (magnesium chloride extractable), (2) acid (hydrochloric acid) soluble, (3) reactive organic-bound (pyrophosphate extractable), (4) crystalline oxide (citrate buffered dithionite (CBD)) extractable, (5) pyrite-bound (nitric acid extractable) and (6) residual (acid/peroxide digestible) forms of iron. Given its intended use for acid sulfate soil materials that frequently contain pyrite, a primary aim of this new sequential extraction procedure was to differentiate iron bound in pyrite from iron contained in other minerals. The results demonstrated that dissolution of pyrite was effectively isolated in the pyrite-bound extraction step, with dissolution of other iron mineral phases (i.e. akaganeite, ferrihydrite, goethite, hematite, jarosite, magnetite, and schwertmannite) occurring within the other five extraction steps. Following a systematic examination of these synthetic iron mineral phases, the sequential extraction scheme was applied to an acid sulfate soil profile, with detailed data presented for two soil layers: one representative of the sulfidic (unoxidised) conditions, and the other sulfuric (oxidised) conditions. Partitioning data for pyrite-bound iron in the acid sulfate soil profile showed good agreement with that calculated via the independently measured pyrite-bound reduced sulfur. This study indicates that the new sequential extraction procedure is suitable for the assessment of iron partitioning in acid sulfate soil materials.