Growth rings in woody plants form in response to seasonal variation in the environment and are fundamental to dendrochronological studies, but estimating plant ages—especially in underexplored growth forms such as forbs, shrubs and subshrubs from grasslands—remains challenging. Here, we address a knowledge gap in the anatomy and histochemistry of subshrubs from natural Cerrado grasslands and evaluate their potential for dendrochronological applications. We studied underground woody organs of Jacaranda decurrens, Lippia lupulina, and Mandevilla longiflora, collected at the Santa Bárbara Ecological Station (Brazil). We used autofluorescence microscopy and a suite of histochemical tests targeting structural and non-structural compounds. Autofluorescence allowed spatial assessment of wood tissues without staining, and improved growth-ring visualization. FASGA staining increased contrast between fibers and parenchyma, facilitating tissue discrimination and growth-ring delimitation, while Mäule staining highlighted differences in cell-wall composition and guaiacyl/syringyl (G/S) ratios throughout growth-ring formation. Starch was consistently detected in parenchymatic cells of all species (lowest in J. decurrens, intermediate in L. lupulina, highest in M. longiflora), and its spatial association with parenchyma aided growth-ring identification. Combining fluorescence and histochemical approaches provides complementary insights into the anatomy and chemistry of underground organs and advances dendrochronological studies in grassland ecosystems.