Background and aims: Rice (Oryza sativa L.) and pearl millet (Pennisetum glaucum L.) biofortification breeding programs require accurate and convenient methods to identify nutrient dense genotypes. The aim of this study was to investigate energy-dispersive X-ray fluorescence spectrometry (EDXRF) for the measurement of zinc (Zn) and iron (Fe) concentration in whole grain rice and pearl millet. Methods: Grain samples were obtained from existing biofortification breeding programs. Reference Zn and Fe concentrations obtained by inductively-coupled plasma-optical emission spectroscopy (ICP-OES) were used to calibrate the EDXRF instrument. Calibration was performed with 24 samples and separate calibrations were developed for rice and pearl millet. To validate calibrations, EDXRF analyses were conducted on an additional 40 samples of each species. Results: EDXRF results were highly correlated with ICP-OES values for both Zn and Fe in both species (r2 = 0. 79 to 0. 98). EDXRF predicted Zn and Fe in rice to within 1. 9 and 1. 6 mg kg-1 of ICP-OES values, and Zn and Fe in pearl millet to within 7. 6 and 12. 5 mg kg-1 of ICP-OES values, at a 95% confidence level. Conclusion: EDXRF offers a convenient, economical tool for screening Zn and Fe concentration in rice and pearl millet biofortification breeding programs.