Soil and plant samples were collected from an ongoing long-term experiment (LTE) at the Indian Agricultural Research Institute farm, New Delhi, to study the distribution of various fractions of iron (Fe) and their contribution to availability and plant uptake in a maize-wheat sequence. The optimum dose-based treatments adopted for the study were nitrogen (N), nitrogen-phosphorus (NP), nitrogen-phosphorus-potassium (NPK), NPK + farmyard manure (FYM), NPK+ zinc (Zn), and control (no fertilizer or manure). Different fractions of Fe in the soil were sequentially extracted using different extractants. Diethylenetriaminepentaacetic acid (DTPA)-extractable Fe did not differ significantly among the treatments as a result of continuous cropping for more than three decades. The overall mean total iron (Fe) content varied from 2.36 to 2.61% under different treatments. Residual Fe constitutes a major portion of total Fe in all four layers of soil. The Fe associated with easily reducible Mn and organic matter contributed directly to DTPA-extractable Fe both in pre-maize and post-wheat soil. Residual Fe contributed directly to uptake Fe by maize and wheat crops.