X-ray fluorescence (XRF) has recently become a powerful tool for the non-destructive elemental analysis of plant materials, providing critical insights into the composition and distribution of major and trace elements. This chapter explores the application of XRF analytical techniques in plant biology studies, particularly emphasizing the use of benchtop μ-XRF systems. Benchtop μ-XRF systems with lateral resolution down to 5 μm offer accessible and efficient elemental analyses that are suitable for routine laboratory studies and elemental screening of plant organs and tissues in environmental, agricultural, and biological studies. On the other hand, synchrotron μ-XRF (SXRFI) with monochromatic excitation provides significantly higher spatial resolution (down to nanometric scale) and sensitivity (down to sub μg g−1 level), enabling detailed mapping of elemental distributions at the cellular and subcellular levels. This capability is essential for studying the mechanisms of nutrient uptake, metal transport, and the localization of potentially toxic elements in plants. The chapter discusses recent advancements in the application of XRF in plant biology with an emphasis on the element distribution in plant organs by benchtop μ-XRF systems. In addition, it addresses the challenges in sample preparation, quantification methods, and the analysis and interpretation of complex data sets.