Maize is a major cereal crop widely consumed in developing countries, which have a high prevalence of iron (Fe) deficiency including anemia. The major cause of Fe deficiency in these countries is inadequate intake of bioavailable Fe, of which poverty is a major contributing factor. Therefore, biofortification of maize has great potential to alleviate this deficiency. Maize is also a model system for genetic and genomic research and thus allows the opportunity for gene discovery. Here we describe an integrated genetic and physiological analysis of Fe nutrition in maize kernels, to determine the genes and molecular processes that influence seed Fe content and bioavailability. Quantitative trait locus (QTL) analysis was used to dissect seed Fe concentration (FeSC) and Fe bioavailability (FeSB) from the Intermated B73 x Mo17 (IBM) recombinant inbred (RI) set of maize. FeSB was determined by an in vitro digestion/Caco-2 cell line bioassay. Loci associated with increased Fe bioavailability were identified on chromosomes 3, 6 and 9 while those associated with increased seed Fe content were identified on chromosomes 1, 2 and 5. Models obtained explained ~25% of the variance in Fe bioavailability and ~20% of the variance in seed Fe content. Seed Fe concentration was not correlated with Fe bioavailability. Iron bioavailability was also not correlated with the levels of seed phytate, as estimated by testing RI at the extremes of the observed Fe bioavailability. FeSB QTL were confirmed using BC3S3 lines derived in both B73 and Mo17 backgrounds.