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Roots are key components of terrestrial ecosystems, yet little is known about how root structure and function vary across a broad range of species, functional groups and ecological gradients in situ.We assessed how woody and grass root anatomical traits vary among soil depths and different fire frequencies to better understand the water‐use strategies exhibited by these two functional groups in tallgrass prairie experiencing woody encroachment. Specifically, we asked: (a) Do root anatomical traits differ with fire frequency or soil depth?; (b) Do relationships between anatomical traits that confer hydraulic safety versus efficiency vary by fire frequency or soil depth?; (c) Is root anatomy associated with integrative root traits (e.g. root diameter, specific root length (SRL) and root biomass)?; and (d) When scaled by root biomass, do root water‐use traits impact the capacity for water uptake?We collected grass and woody roots from 10, 30 and 50 cm deep soil in areas burned every 1, 4 and 20 years. We then measured xylem conduit diameter, conduit cell wall thickness, conduit number, conduit mechanical safety (t/b), stele area, endoderm thickness, hydraulic diameter, theoretical hydraulic conductivity and root‐system theoretical hydraulic conductance.We observed: (a) Woody roots had high hydraulic conductance in shallow soils and greater mechanical strength in deeper soils, which may provide a competitive advantage in less frequently burned, more diverse plant communities; (b) shallow grass roots had unique trait combinations at the anatomical and root‐system levels (thinner, more numerous conduits and higher root‐system hydraulic conductance compared to deeper roots) that likely allow these plants to rapidly use water but tolerate dry soils under multiple fire regimes; and (c) hydraulic safety versus efficiency trade‐offs translate between different hierarchical scales (i.e. from anatomical to integrative root traits).These results provide anatomical evidence to explain water‐use dynamics in tallgrass prairie and also provide novel insight regarding functional strategies that may facilitate the conversion from grassland to shrubland in less frequently burned tallgrass prairie. Future work should investigate these dynamics in situ, as they may explain current and future patterns of woody‐grass coexistence in tallgrass prairies. A free Plain Language Summary can be found within the Supporting Information of this article. [ABSTRACT FROM AUTHOR]