Background and aims: Crop biofortification programs require fast, accurate and inexpensive methods of identifying nutrient dense genotypes. This study investigated energy-dispersive X-ray fluorescence spectrometry (EDXRF) for the measurement of zinc (Zn), iron (Fe) and selenium (Se) concentrations in whole grain wheat. Methods: Grain samples were obtained from existing biofortification programs. Reference Zn, Fe and Se concentrations were obtained using inductively coupled plasma optical emission spectrometry (ICP-OES) and/or inductively coupled plasma mass spectrometry (ICP-MS). One set of 25 samples was used to calibrate for Zn (19-60 mg kg-1) and Fe (26-41 mg kg-1), with 25 further samples used to calibrate for Se (2-31 mg kg-1). Calibrations were validated using an additional 40-50 wheat samples. Results: EDXRF limits of quantification (LOQ) were estimated as 7, 3 and 2 mg kg-1 for Zn, Fe, and Se, respectively. EDXRF results were highly correlated with ICP-OES or -MS values. Standard errors of EDXRF predictions were ±2. 2 mg Zn kg-1, ±2. 6 mg Fe kg-1, and ±1. 5 mg Se kg-1. Conclusion: EDXRF offers a fast and economical method for the assessment of Zn, Fe and Se concentration in wheat biofortification programs.