<p>Rutaceae plants are a rich source of alkaloids, but their cell-specific localization remains relatively unknown in the underground tissues, except for the acridone-containing idioblasts in <i>Ruta graveolens</i> roots. To fill this gap, we used broad taxonomic sampling to investigate both the microchemistry and the root structure of Rutaceae species. Utilizing both transmitted light and epifluorescence microscopy, we examined seven distinct Rutaceae species to determine if root idioblasts were present. For the microchemical analysis of the idioblasts contents, we employed Nile red staining and Dragendorff’s test. All analyzed species presented root idioblasts with contents that are reactive with Dragendorff’s reagent and Nile red, suggesting the presence of putative alkaloids and lipophilic environments, respectively. These idioblasts are typically found in the radicular cortex and can be easily observed under UV light due to their autofluorescent contents. We found that the occurrence of root idioblasts is a widely distributed condition in Rutaceae, showing for the first time both structural and microchemical analyses for broad taxonomic sampling. These cell types could play an important role in alkaloid metabolism in the underground tissues of plants in this family and offer an exciting opportunity for future investigations.</p>