This study aimed to determine differences among wheat cultivars in the distribution and speciation of Fe and Zn in grain milling fractions. Cultivars with higher Fe and Zn concentrations in the wholemeal flour were found to contain higher concentrations in the white flour. Soluble Fe and Zn were extracted and analyzed by size exclusion-inductively coupled plasma mass spectrometry. Fe speciation varied between milling fractions with a low molecular weight (LMW) complex likely to be Fe-deoxymugenic acid/nicotianamine being the predominant extractable Fe species in white flour, accounting for approximately 85% of the extractable Fe. Bran fractions had a lower amount of LMW-Fe form but more as soluble Fe-phytate and an unidentified high molecular weight peak. In the white flour fraction soluble Zn was found to be present mainly as a LMW peak likely to be Zn-nicotianamine complex. Soluble Fe-phytate was found in the white flour fraction of a high-Fe cultivar but not in a low-Fe cultivar.