A coupled in vitro digestion/Caco-2 model was employed to assess iron bioavailability from wheat Aegilops derivatives selected for high iron and protein contents. The iron content in wheat genotypes used in this study correlated to a great extent with both protein (r = 0.80) and phytate (r = 0.68) contents. The iron bioavailability was based on Caco-2 cell ferritin formation from cooked digests of these derivatives (relative to WL711 control) and correlated positively with dialyzable iron (r = 0.63) and total iron content (r = 0.38) but not with the phytate content. The apparently decreased phytate/iron molar ratios, however, correlated negatively (r = -0.42) with the iron bioavailability, justifying the utilization of these parameters in biofortification programs. Iron bioavailability in the derivatives increased up to 1.5-fold, corresponding to a 1.5-2.2-fold increase observed in iron content over control. These data suggest that biofortification for iron proportionately leading to higher iron bioavailability will be the most feasible and cost-effective approach to combat micronutrient deficiency.