Iron (Fe) deficiency is the most prevalent nutrient deficiency worldwide. Biofortification of staple food crops, such as the lentil (Lens culinaris L.), may be an effective solution. We analyzed the Fe concentration, Fe bioavailability, and phytic acid (PA) concentration of 23 lentil genotypes grown in five different locations in Saskatchewan, Canada. Relative Fe bioavailability, assessed using the in vitro digestion/Caco-2 cell model, varied significantly among the genotypes. Significant location effects were observed for Fe concentration, relative bioavailability, and PA concentration. Associations were observed between Fe concentration and PA concentration (r=0.55, p<0.001), Fe concentration and relative Fe bioavailability (r=−0.41, p<0.001), and Fe bioavailability and phytate content (r=−0.78, p<0.001). These results demonstrate significant biofortification potential, and establish a baseline for relative Fe bioavailability in the lentil.